Strand displacing amplification enzymes

ABSTRACT

Disclosed herein, inter alia, are novel strand-displacing polymerases and methods of use thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/292,890 filed on Dec. 22, 2021, which is incorporated herein byreference in its entirety and for all purposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The Sequence Listing titled 051385-564001US_SL_ST26.XML, was created onDec. 15, 2022 in machine format IBM-PC, MS-Windows operating system, is21,959 bytes in size, and is hereby incorporated by reference in itsentirety for all purposes.

BACKGROUND

Genetic analysis is taking on increasing importance in modern society asa diagnostic, prognostic, and as a forensic tool, and typically requiresamplification of genomic fragments. A majority of nucleic acidamplification techniques (e.g., DNA amplification) used in university,medical, and clinical laboratory research is performed using thepolymerase chain reaction (PCR), though in the past decade alternativeamplification methods have emerged that eliminate thermal cycling (i.e.,isothermal amplification). Typical isothermal amplification methodsrequire the use of a DNA polymerase with a strong strand displacementactivity to displace downstream DNA, thereby enabling continuousreplication without thermal cycling. Efficient amplification typicallyrequires elevated temperatures to enable the annealing of primers atspecific locations on the dsDNA. However, few thermostable stranddisplacing enzymes exist. For example, SD DNA polymerase (a mutant TaqDNA polymerase) and the large fragment of Bst DNA polymerase possessfavorable characteristics for isothermal amplification, but both areinactivated and elevated temperatures (e.g., greater than 70° C.). Thus,there is a need for thermostable, strand-displacing polymerases.Disclosed herein, inter alia, are solutions to these and other problemsin the art.

BRIEF SUMMARY

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including a first mutation at amino acid position588 or an amino acid position corresponding to position 588; wherein thefirst mutation is leucine, isoleucine, valine, alanine, or glycine; anda second mutation at amino acid position 7 or an amino acid positioncorresponding to position 7, wherein the second mutation is histidine,lysine, or arginine; a second mutation at amino acid position 97 or anamino acid position corresponding to position 97, wherein the secondmutation is cysteine, histidine, lysine, serine, threonine, ormethionine; or a second mutation at amino acid position 742 or an aminoacid position corresponding to position 742, wherein the second mutationis leucine, isoleucine, alanine, or glycine.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including a first mutation at amino acid position97 or an amino acid position corresponding to position 97, wherein thefirst mutation includes cysteine, histidine, lysine, serine, threonine,or methionine; a second mutation at amino acid position 588 or an aminoacid position corresponding to position 588; wherein the second mutationincludes leucine, isoleucine, valine, alanine, or glycine.

In an aspect is provided a method of incorporating a nucleotide into anucleic acid sequence including combining in a reaction vessel: (i) anucleic acid template, (ii) a nucleotide solution, and (iii) apolymerase, wherein the polymerase is a polymerase as described herein.In embodiments, the method includes combining the components in areaction vessel under conditions for incorporating and/orpolymerization. Such conditions are known in the art and describedherein.

In another aspect is provided a method of amplifying a nucleic acidsequence including: a. hybridizing a nucleic acid template with a primerto form a primer-template hybridization complex; b. contacting theprimer-template hybridization complex with a DNA polymerase andnucleotides, wherein the DNA polymerase is the polymerase as describedherein; and c. subjecting the primer-template hybridization complex toconditions which enable the polymerase to incorporate one or morenucleotides into the primer-template hybridization complex to generateamplification products, thereby amplifying a nucleic acid sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an alignment of two sequences described herein. SEQ IDNO:3 is aligned to SEQ ID NO:1 and amino acid positions 541-560 aredepicted in FIG. 1 . The alignment highlights a deletion in SEQ ID NO:3relative to SEQ ID NO:1, such that any amino acid positions beyond aminoacid position 554 are shifted −1 in SEQ ID NO:3 relative to SEQ ID NO:1(i.e., amino acid E554 in SEQ ID NO:3 corresponds to E555 in SEQ IDNO:1).

DETAILED DESCRIPTION

The aspects and embodiments described herein relate to strand displacingpolymerases and uses thereof.

Definitions

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

“Nucleic acid” refers to nucleotides (e.g., deoxyribonucleotides orribonucleotides) and polymers thereof in either single-, double- ormultiple-stranded form, or complements thereof. The terms“polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in theusual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

The term “duplex” in the context of polynucleotides refers, in the usualand customary sense, to double strandedness. Nucleic acids can be linearor branched. For example, nucleic acids can be a linear chain ofnucleotides or the nucleic acids can be branched, e.g., such that thenucleic acids comprise one or more arms or branches of nucleotides.Optionally, the branched nucleic acids are repetitively branched to formhigher ordered structures such as dendrimers and the like. Differentpolynucleotides may have different three-dimensional structures, and mayperform various functions, known or unknown.

Nucleic acids, including e.g., nucleic acids with a phosphothioatebackbone, can include one or more reactive moieties. As used herein, theterm reactive moiety includes any group capable of reacting with anothermolecule, e.g., a nucleic acid or polypeptide through covalent,non-covalent or other interactions. By way of example, the nucleic acidcan include an amino acid reactive moiety that reacts with an amino acidon a protein or polypeptide through a covalent, non-covalent or otherinteraction.

The term “base” and “nucleobase” as used herein refers to a purine orpyrimidine compound, or a derivative thereof, that may be a constituentof nucleic acid (i.e. DNA or RNA, or a derivative thereof). Inembodiments, the base is a derivative of a naturally occurring DNA orRNA base (e.g., a base analogue). In embodiments, the base is abase-pairing base. In embodiments, the base pairs to a complementarybase. In embodiments, the base is capable of forming at least onehydrogen bond with a complementary base (e.g., adenine hydrogen bondswith thymine, adenine hydrogen bonds with uracil, guanine pairs withcytosine). Non-limiting examples of a base includes cytosine or aderivative thereof (e.g., cytosine analogue), guanine or a derivativethereof (e.g., guanine analogue), adenine or a derivative thereof (e.g.,adenine analogue), thymine or a derivative thereof (e.g., thymineanalogue), uracil or a derivative thereof (e.g., uracil analogue),hypoxanthine or a derivative thereof (e.g., hypoxanthine analogue),xanthine or a derivative thereof (e.g., xanthine analogue), guanosine ora derivative thereof (e.g., 7-methylguanosine analogue), deaza-adenineor a derivative thereof (e.g., deaza-adenine analogue), deaza-guanine ora derivative thereof (e.g., deaza-guanine), deaza-hypoxanthine or aderivative thereof, 5,6-dihydrouracil or a derivative thereof (e.g.,5,6-dihydrouracil analogue), 5-methylcytosine or a derivative thereof(e.g., 5-methylcytosine analogue), or 5-hydroxymethylcytosine or aderivative thereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is thymine, cytosine, uracil, adenine, guanine,hypoxanthine, xanthine, theobromine, caffeine, uric acid, or isoguanine.In embodiments, the base is

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry or anaqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The terms “analog” and “analogue” and “derivative” in reference to achemical compound, refers to compounds having a structure similar tothat of another one, but differing from it in respect of one or moredifferent atoms, functional groups, or substructures that are replacedwith one or more other atoms, functional groups, or substructures. Inthe context of a nucleotide useful in practicing the invention, anucleotide analog refers to a compound that, like the nucleotide ofwhich it is an analog, can be incorporated into a nucleic acid molecule(e.g., an extension product) by a suitable polymerase, for example, aDNA polymerase in the context of a dNTP analogue. The terms alsoencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs include,include, without limitation, phosphodiester derivatives including, e.g.,phosphoramidate, phosphorodiamidate, phosphorothioate (also known asphosphothioate having double bonded sulfur replacing oxygen in thephosphate), phosphorodithioate, phosphonocarboxylic acids,phosphonocarboxylates, phosphonoacetic acid, phosphonoformic acid,methyl phosphonate, boron phosphonate, or O-methylphosphoroamiditelinkages (see Eckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICALAPPROACH, Oxford University Press) as well as modifications to thenucleotide bases such as in 5-methyl cytidine or pseudouridine; andpeptide nucleic acid backbones and linkages. Other analog nucleic acidsinclude those with positive backbones; non-ionic backbones, modifiedsugars, and non-ribose backbones (e.g. phosphorodiamidate morpholinooligos or locked nucleic acids (LNA) as known in the art), includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS INANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one ormore carbocyclic sugars are also included within one definition ofnucleic acids. Modifications of the ribose-phosphate backbone may bedone for a variety of reasons, e.g., to increase the stability andhalf-life of such molecules in physiological environments or as probeson a biochip. Mixtures of naturally occurring nucleic acids and analogscan be made; alternatively, mixtures of different nucleic acid analogs,and mixtures of naturally occurring nucleic acids and analogs may bemade. In embodiments, the internucleotide linkages in DNA arephosphodiester, phosphodiester derivatives, or a combination of both.

The term “complement,” as used herein, refers to a nucleotide (e.g., RNAor DNA) or a sequence of nucleotides capable of base pairing with acomplementary nucleotide or sequence of nucleotides. As described hereinand commonly known in the art, the complementary (matching) nucleosideof adenosine is thymidine and the complementary (matching) nucleoside ofguanosine is cytidine. Thus, a complement may include a sequence ofnucleotides that base pair with corresponding complementary nucleotidesof a second nucleic acid sequence. The nucleotides of a complement maymatch, partially or completely, the nucleotides of the second nucleicacid sequence. Where the nucleotides of the complement completely matcheach nucleotide of the second nucleic acid sequence, the complementforms base pairs with each nucleotide of the second nucleic acidsequence. Where the nucleotides of the complement partially match thenucleotides of the second nucleic acid sequence, only some of thenucleotides of the complement form base pairs with nucleotides of thesecond nucleic acid sequence. Examples of complementary sequencesinclude coding and non-coding sequences, wherein the non-coding sequencecontains complementary nucleotides to the coding sequence and thus formsthe complement of the coding sequence. A further example ofcomplementary sequences are sense and antisense sequences, wherein thesense sequence contains complementary nucleotides to the antisensesequence and thus forms the complement of the antisense sequence.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other may have aspecified percentage of nucleotides that are complementary (i.e., about60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion).

“DNA” refers to deoxyribonucleic acid, a polymer of deoxyribonucleotides(e.g., dATP, dCTP, dGTP, dTTP, dUTP, etc.) linked by phosphodiesterbonds. DNA can be single-stranded (ssDNA) or double-stranded (dsDNA),and can include both single and double-stranded (or “duplex”) regions.“RNA” refers to ribonucleic acid, a polymer of ribonucleotides linked byphosphodiester bonds. RNA can be single-stranded (ssRNA) ordouble-stranded (dsRNA), and can include both single and double-stranded(or “duplex”) regions. Single-stranded DNA (or regions thereof) andssRNA can, if sufficiently complementary, hybridize to formdouble-stranded DNA/RNA complexes (or regions).

The term “DNA primer” refers to any DNA molecule that may hybridize to aDNA template and be bound by a DNA polymerase and extended in atemplate-directed process for nucleic acid synthesis. The term “DNAtemplate” refers to any DNA molecule that may be bound by a DNApolymerase and utilized as a template for nucleic acid synthesis.

The term “dATP analogue” refers to an analogue of deoxyadenosinetriphosphate (dATP) that is a substrate for a DNA polymerase. The term“dCTP analogue” refers to an analogue of deoxycytidine triphosphate(dCTP) that is a substrate for a DNA polymerase. The term “dGTPanalogue” refers to an analogue of deoxyguanosine triphosphate (dGTP)that is a substrate for a DNA polymerase. The term “dNTP analogue”refers to an analogue of deoxynucleoside triphosphate (dNTP) that is asubstrate for a DNA polymerase. The term “dTTP analogue” refers to ananalogue of deoxythymidine triphosphate (dUTP) that is a substrate for aDNA polymerase. The term “dUTP analogue” refers to an analogue ofdeoxyuridine triphosphate (dUTP) that is a substrate for a DNApolymerase.

The term “extendible” means, in the context of a nucleotide, primer, orextension product, that the 3′-OH group of the particular molecule isavailable and accessible to a DNA polymerase for extension or additionof nucleotides derived from dNTPs or dNTP analogues. “Incorporation”means joining of the modified nucleotide to the free 3′ hydroxyl groupof a second nucleotide via formation of a phosphodiester linkage withthe 5′ phosphate group of the modified nucleotide. The second nucleotideto which the modified nucleotide is joined will typically occur at the3′ end of a polynucleotide chain. As used herein, the term“incorporating” or “chemically incorporating,” when used in reference toa primer and a nucleotide, refers to the process of joining thenucleotide to the primer or extension product thereof by formation of aphosphodiester bond. As used herein, the term “extension” or“elongation” is used in accordance with their plain and ordinarymeanings and refer to synthesis by a polymerase of a new polynucleotidestrand (i.e., an “extension strand”) complementary to a template strandby adding free nucleotides (e.g., dNTPs) from a reaction mixture thatare complementary to the template in a 5′-to-3′ direction, includingcondensing a 5′-phosphate group of a dNTPs with a 3′-hydroxy group atthe end of the nascent (elongating) DNA strand.

Descriptions of nucleotide analogues of the present disclosure arelimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics,which are not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may in embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein, which encodes apolypeptide, also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid that encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosure.

The following groups each contain amino acids that are conservativesubstitutions for one another: 1) Non-polar—Alanine (A), Leucine (L),Isoleucine (I), Valine (V), Glycine (G), Methionine (M); 2)Aliphatic—Alanine (A), Leucine (L), Isoleucine (I), Valine (V); 3)Acidic—Aspartic acid (D), Glutamic acid (E); 4) Polar—Asparagine (N),Glutamine (Q); Serine (S), Threonine (T); 5) Basic—Arginine (R), Lysine(K); 7) Aromatic—Phenylalanine (F), Tyrosine (Y), Tryptophan (W),Histidine (H); 8) Other—Cysteine (C) and Proline (P).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithm with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the complement of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions. As described below,the preferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. As used herein, percent (%) aminoacid sequence identity is defined as the percentage of amino acids in acandidate sequence that is identical to the amino acids in a referencesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent sequence identity can be achieved invarious ways that are within the level of skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full length of the sequences beingcompared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 700, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The term “DNA polymerase” and “nucleic acid polymerase” are used inaccordance with their plain ordinary meaning and refer to enzymescapable of synthesizing nucleic acid molecules from nucleotides (e.g.,deoxyribonucleotides). Typically, a DNA polymerase adds nucleotides tothe 3′-end of a DNA strand, one nucleotide at a time. In embodiments,the DNA polymerase is a Pol I DNA polymerase, Pol II DNA polymerase, PolIII DNA polymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol βDNA polymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNApolymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNApolymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNApolymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNApolymerase, Pol υ DNA polymerase, or a thermophilic nucleic acidpolymerase (e.g. Therminator γ, 9° N polymerase (exo-), Therminator II,Therminator III, or Therminator IX). In embodiments, the DNA polymeraseis a Pyrococcus DNA polymerase. For example, a polymerase catalyzes theaddition of a next correct nucleotide to the 3′-OH group of the primervia a phosphodiester bond, thereby chemically incorporating thenucleotide into the primer. Optionally, the polymerase used in theprovided methods is a processive polymerase. Optionally, the polymeraseused in the provided methods is a distributive polymerase.

The term “thermophilic nucleic acid polymerase” as used herein refers toa family of DNA polymerases (e.g., 9° N™) and mutants thereof derivedfrom the DNA polymerase originally isolated from the hyperthermophilicarchaea, Thermococcus sp. 9 degrees N-7, found in hydrothermal vents atthat latitude (East Pacific Rise) (Southworth M W, et al. PNAS. 1996;93(11):5281-5285). A thermophilic nucleic acid polymerase is a member ofthe family B DNA polymerases. Site-directed mutagenesis of the 3′-5′ exomotif I (Asp-Ile-Glu or DIE) to AIA, AIE, EIE, EID or DIA yieldedpolymerase with no detectable 3′ exonuclease activity. Mutation toAsp-Ile-Asp (DID) resulted in reduction of 3′-5′ exonuclease specificactivity to <1% of wild type, while maintaining other properties of thepolymerase, including its high strand displacement activity. Thesequence AIA (D141A, E143A) was chosen for reducing exonuclease.Subsequent mutagenesis of key amino acids results in an increasedability of the enzyme to incorporate dideoxynucleotides, ribonucleotidesand acyclonucleotides (e.g., Therminator II enzyme from New EnglandBiolabs with D141A/E143A/Y409V/A485L mutations); 3′-amino-dNTPs,3′-azido-dNTPs and other 3′-modified nucleotides (e.g., NEB TherminatorIII DNA Polymerase with D141A/E143A/L4085/Y409A/P410V mutations, NEBTherminator IX DNA polymerase), or γ-phosphate labeled nucleotides(e.g., Therminator γ:D141A/E143A/W355A/L408W/R460A/Q461S/K464E/D480V/R484W/A485L). Typically,these enzymes do not have 5′-3′ exonuclease activity. Additionalinformation about thermophilic nucleic acid polymerases may be found in(Southworth M W, et al. PNAS. 1996; 93(11):5281-5285; Bergen K, et al.ChemBioChem. 2013; 14(9):1058-1062; Kumar S, et al. Scientific Reports.2012; 2:684; Fuller C W, et al. 2016; 113(19):5233-5238; Guo J, et al.PNAS. 2008; 105(27):9145-9150), which are incorporated herein in theirentirety for all purposes.

In the context of this application, the term “motif A region”specifically refers to the three amino acids functionally equivalent,corresponding to, positionally equivalent, or homologous to amino acids409, 410, and 411 in wild type P. horikoshii; these amino acids arefunctionally equivalent to amino acid positions 408, 409, and 410 in 9°N polymerase. Functionally equivalent, positionally equivalent, orhomologous “motif A regions” of polymerases other than P. horikoshii canbe identified on the basis of amino acid sequence alignment and/ormolecular modeling. Sequence alignments may be compiled using any of thestandard alignment tools known in the art, such as for example BLAST,DIAMOND (Buchfink et al. Nat Methods 12, 59-60 (2015)), and the like.

The terms “position”, “numbered with reference to” or “correspondingto,” when used in the context of the numbering of a given amino acid orpolynucleotide sequence, refer to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. Similarly, the term“functionally equivalent to” in relation to an amino acid positionrefers to an amino acid residue in a protein that corresponds to aparticular amino acid in a reference sequence. An amino acid“corresponds” to a given residue when it occupies the same essentialstructural position within the protein as the given residue. One skilledin the art will immediately recognize the identity and location ofresidues corresponding to a specific position in a protein (e.g.,polymerase) in other proteins with different numbering systems. Forexample, by performing a simple sequence alignment with a protein (e.g.,polymerase) the identity and location of residues corresponding tospecific positions of said protein are identified in other proteinsequences aligning to said protein. For example, a selected residue in aselected protein corresponds to methionine at position 129 when theselected residue occupies the same essential spatial or other structuralrelationship as a methionine at position 129. In some embodiments, wherea selected protein is aligned for maximum homology with a protein, theposition in the aligned selected protein aligning with methionine 129 issaid to correspond to methionine 129. Instead of a primary sequencealignment, a three-dimensional structural alignment can also be used,e.g., where the structure of the selected protein is aligned for maximumcorrespondence with the methionine at position 129, and the overallstructures compared. In this case, an amino acid that occupies the sameessential position as methionine 129 in the structural model is said tocorrespond to the methionine 129 residue. For example, references to aP. horikoshii polymerase amino acid position recited herein may refer toa numbered position set forth in SEQ ID NO:1, or the correspondingposition in a polymerase homolog of SEQ ID NO:1. For example, whenidentifying an amino acid that corresponds to a position in SEQ ID NO:1,aligning the second enzyme species identifies if any insertions and/ordeletions shift the position of the amino acid. An alignment of SEQ IDNO:3 to SEQ ID NO:1, a portion of which is provided in FIG. 1 ,highlights a deletion in SEQ ID NO:3 relative to SEQ ID NO:1, such thatany amino acid positions beyond amino acid position 554 are shifted −1in SEQ ID NO:3 relative to SEQ ID NO:1 (i.e., amino acid E554 in SEQ IDNO:3 corresponds to E555 in SEQ ID NO:1).

In embodiments, the polymerase may include an amino acid substitutionmutation at a particular position corresponding to a position in SEQ IDNO: 1. For example, in embodiments, the polymerase includes an aminoacid substitution mutation at position 141, which means the variantpolymerase has a different amino acid at position 141 compared to SEQ IDNO: 1. In embodiments, the polymerase includes an amino acidsubstitution mutation at more than one position compared to SEQ IDNO: 1. For example, in embodiments, the polymerase includes thefollowing substitution mutations: D141A; E143A; L4095; Y410A; P411V,where the number refers to the corresponding position in SEQ ID NO: 1.One having skill in the art would understand the amino acid mutationnomenclature, such that D141A refers to aspartic acid (single lettercode is D), at position 141, being replaced with alanine (single lettercode A).

The term “exonuclease activity” is used in accordance with its ordinarymeaning in the art, and refers to the removal of a nucleotide from anucleic acid by a DNA polymerase. For example, during polymerization,nucleotides are added to the 3′ end of the primer strand. Occasionally aDNA polymerase incorporates an incorrect nucleotide to the 3′-OHterminus of the primer strand, wherein the incorrect nucleotide cannotform a hydrogen bond to the corresponding base in the template strand.Such a nucleotide, added in error, is removed from the primer as aresult of the 3′ to 5′ exonuclease activity of the DNA polymerase. Inembodiments, exonuclease activity may be referred to as “proofreading.”When referring to 3′-5′ exonuclease activity, it is understood that theDNA polymerase facilitates a hydrolyzing reaction that breaksphosphodiester bonds at the 3′ end of a polynucleotide chain to excisethe nucleotide, thereby releasing deoxyribonucleoside 5′-monophosphatesone after another. One having skill in the art understands that anenzyme having 3′-5′ exonuclease activity does not cleave DNA strandswithout terminal 3′-OH moieties. In embodiments, 3′-5′ exonucleaseactivity refers to the successive removal of nucleotides insingle-stranded DNA in a 3′→5′ direction, releasing deoxyribonucleoside5′-monophosphates one after another. Methods for quantifying exonucleaseactivity are known in the art, see for example Southworth et al, PNASVol 93, 8281-8285 (1996).

The terms “measure”, “measuring”, “measurement” and the like refer notonly to quantitative measurement of a particular variable, but also toqualitative and semi-quantitative measurements. Accordingly,“measurement” also includes detection, meaning that merely detecting achange, without quantification, constitutes measurement.

A “polymerase-template complex” refers to a functional complex between aDNA polymerase and a DNA primer-template molecule (e.g., nucleic acid).In embodiments, the polymerase is non-covalently bound to a nucleic acidprimer and the template nucleic acid molecule.

The term “solid substrate” means any suitable medium present in thesolid phase to which an antibody or an agent can be covalently ornon-covalently affixed or immobilized. Preferred solid substrates areglass. Non-limiting examples include chips, beads and columns. The solidsubstrate can be non-porous or porous. Exemplary solid substratesinclude, but are not limited to, glass and modified or functionalizedglass, plastics (including acrylics, polystyrene and copolymers ofstyrene and other materials, polypropylene, polyethylene, polybutylene,polyurethanes, Teflon™, cyclic olefins, polyimides, etc.), nylon,ceramics, resins, Zeonor, silica or silica-based materials includingsilicon and modified silicon, carbon, metals, inorganic glasses, opticalfiber bundles, and polymers.

The term “species”, when used in the context of describing a particularcompound or molecule species, refers to a population of chemicallyindistinct molecules. When used in the context of taxonomy, “species” isthe basic unit of classification and a taxonomic rank. For example, inreference to the microorganism Pyrococcus horikoshii, horikoshii is aspecies of the genus Pyrococcus.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g., ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects(e.g., enzymes) or reagents of the experiment are treated as in aparallel experiment except for omission of a procedure, reagent, orvariable of the experiment (e.g., a polymerase not having one or moremutations relative to the polymerase being tested). In some instances,the control is used as a standard of comparison in evaluatingexperimental effects. In some embodiments, a control is the measurementof the activity of a protein in the absence of a mutation as describedherein (including embodiments and examples). “Control polymerase” isdefined herein as the polymerase against which the activity of thealtered polymerase is compared. In one embodiment of the invention thecontrol polymerase may comprise a wild type polymerase or an exo-variantthereof. Unless otherwise stated, by “wild type” it is generally meantthat the polymerase comprises its natural amino acid sequence, as itwould be found in nature. The invention is not limited to merely acomparison of activity of the polymerases as described herein againstthe wild type equivalent or exo-variant of the polymerase that is beingaltered. Many polymerases exist whose amino acid sequence has beenmodified (e.g., by amino acid substitution mutations) and which canprove to be a suitable control for use in assessing the modifiednucleotide incorporation efficiencies of the polymerases as describedherein. The control polymerase can, therefore, comprise any knownpolymerase, including mutant polymerases known in the art. The activityof the chosen “control” polymerase with respect to incorporation of thedesired nucleotide analogues may be determined by an incorporationassay.

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties.

The term “kit” is used in accordance with its plain ordinary meaning andrefers to any delivery system for delivering materials or reagents forcarrying out a method of the invention. Such delivery systems includesystems that allow for the storage, transport, or delivery of reactionreagents (e.g., nucleotides, enzymes, nucleic acid templates, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the reaction, etc.) from onelocation to another location. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme, while a second container containsnucleotides. In embodiments, the kit includes vessels containing one ormore enzymes, primers, adapters, or other reagents as described herein.Vessels may include any structure capable of supporting or containing aliquid or solid material and may include tubes, vials, jars, containers,tips, etc. In embodiments, a wall of a vessel may permit thetransmission of light through the wall. In embodiments, the vessel maybe optically clear. The kit may include the enzyme and/or nucleotides ina buffer. In embodiments, the buffer includes an acetate buffer,3-(N-morpholino)propanesulfonic acid (MOPS) buffer,N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer,phosphate-buffered saline (PBS) buffer,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer,N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(AMPSO) buffer, borate buffer (e.g., borate buffered saline, sodiumborate buffer, boric acid buffer), 2-Amino-2-methyl-1,3-propanediol(AMPD) buffer, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid(CAPSO) buffer, 2-Amino-2-methyl-1-propanol (AMP) buffer,4-(Cyclohexylamino)-1-butanesulfonic acid (CABS) buffer, glycine-NaOHbuffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer,tris(hydroxymethyl)aminomethane (Tris) buffer, or aN-cyclohexyl-3-aminopropanesulfonic acid (CAPS) buffer. In embodiments,the buffer is a borate buffer. In embodiments, the buffer is a CHESbuffer.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The phrase “stringent hybridization conditions” refers to conditionsunder which a primer will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acids, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. For selective or specific hybridization, a positive signal isat least two times background, preferably 10 times backgroundhybridization. Exemplary stringent hybridization conditions can be asfollowing: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C.

“Synthetic” DNA polymerases refer to non-naturally occurring DNApolymerases such as those constructed by synthetic methods, mutatedparent DNA polymerases such as truncated DNA polymerases and fusion DNApolymerases (e.g., as described in U.S. Pat. No. 7,541,170). Variants ofthe parent DNA polymerase have been engineered by mutating residuesusing site-directed or random mutagenesis methods known in the art. Inembodiments, the mutations are in any of Motifs I-VI. The variant isexpressed in an expression system such as E. coli by methods known inthe art. The variant is then screened using the assays described hereinto determine activity (e.g., strand-displacing activity).

As used herein, the term “template polynucleotide” or “template nucleicacid” refers to any polynucleotide molecule that may be bound by apolymerase and utilized as a template for nucleic acid synthesis. Atemplate polynucleotide may be a target polynucleotide. In general, theterm “target polynucleotide” refers to a nucleic acid molecule orpolynucleotide in a starting population of nucleic acid molecules havinga target sequence whose presence, amount, and/or nucleotide sequence, orchanges in one or more of these, are desired to be determined. Ingeneral, the term “target sequence” refers to a nucleic acid sequence ona single strand of nucleic acid. The terms “single strand” and “ssDNA”are used in accordance with its plain and ordinary meaning and refer toa single-stranded polynucleotide. The target sequence may be a portionof a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA,miRNA, rRNA, or others. The target sequence may be a target sequencefrom a sample or a secondary target such as a product of anamplification reaction. A target polynucleotide is not necessarily anysingle molecule or sequence. For example, a target polynucleotide may beany one of a plurality of target polynucleotides in a reaction, or allpolynucleotides in a given reaction, depending on the reactionconditions. For example, in a nucleic acid amplification reaction withrandom primers, all polynucleotides in a reaction may be amplified. As afurther example, a collection of targets may be simultaneously assayedusing polynucleotide primers directed to a plurality of targets in asingle reaction. As yet another example, all or a subset ofpolynucleotides in a sample may be modified by the addition of aprimer-binding sequence (such as by the ligation of adapters containingthe primer binding sequence), rendering each modified polynucleotide atarget polynucleotide in a reaction with the corresponding primerpolynucleotide(s). In the context of selective sequencing, “targetpolynucleotide(s)” refers to the subset of polynucleotide(s) to besequenced from within a starting population of polynucleotides.

In embodiments, a target polynucleotide is a cell-free polynucleotide.In general, the terms “cell-free,” “circulating,” and “extracellular” asapplied to polynucleotides (e.g. “cell-free DNA” (cfDNA) and “cell-freeRNA” (cfRNA)) are used interchangeably to refer to polynucleotidespresent in a sample from a subject or portion thereof that can beisolated or otherwise manipulated without applying a lysis step to thesample as originally collected (e.g., as in extraction from cells orviruses). Cell-free polynucleotides are thus unencapsulated or “free”from the cells or viruses from which they originate, even before asample of the subject is collected. Cell-free polynucleotides may beproduced as a byproduct of cell death (e.g. apoptosis or necrosis) orcell shedding, releasing polynucleotides into surrounding body fluids orinto circulation. Accordingly, cell-free polynucleotides may be isolatedfrom a non-cellular fraction of blood (e.g. serum or plasma), from otherbodily fluids (e.g. urine), or from non-cellular fractions of othertypes of samples.

As used herein, a “native” nucleotide is used in accordance with itsplain and ordinary meaning and refers to a naturally occurringnucleotide that does not include an exogenous label (e.g., a fluorescentdye, or other label) or chemical modification such as those that maycharacterize a nucleotide analog (e.g., a reversible terminatingmoiety). Examples of native nucleotides useful for carrying outprocedures described herein include: dATP(2′-deoxyadenosine-5′-triphosphate); dGTP(2′-deoxyguanosine-5′-triphosphate); dCTP(2′-deoxycytidine-5′-triphosphate); dTTP(2′-deoxythymidine-5′-triphosphate); and dUTP(2′-deoxyuridine-5′-triphosphate). A “canonical” nucleotide is anunmodified nucleotide.

As used herein, the term “modified nucleotide” refers to nucleotidemodified in some manner. Typically, a nucleotide contains a single5-carbon sugar moiety, a single nitrogenous base moiety and 1 to threephosphate moieties. In embodiments, a nucleotide can include a blockingmoiety (alternatively referred to herein as a reversible terminatormoiety) and/or a label moiety. A blocking moiety on a nucleotideprevents formation of a covalent bond between the 3′ hydroxyl moiety ofthe nucleotide and the 5′ phosphate of another nucleotide. A blockingmoiety on a nucleotide can be reversible, whereby the blocking moietycan be removed or modified to allow the 3′ hydroxyl to form a covalentbond with the 5′ phosphate of another nucleotide. A blocking moiety canbe effectively irreversible under particular conditions used in a methodset forth herein. In embodiments, the blocking moiety is attached to the3′ oxygen of the nucleotide and is independently —NH₂, —CN, —CH₃, C₂-C₆allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃), or —CH₂N₃.In embodiments, the blocking moiety is attached to the 3′ oxygen of thenucleotide and is independently

A label moiety of a nucleotide can be any moiety that allows thenucleotide to be detected, for example, using a spectroscopic method.Exemplary label moieties are fluorescent labels, mass labels,chemiluminescent labels, electrochemical labels, detectable labels andthe like. One or more of the above moieties can be absent from anucleotide used in the methods and compositions set forth herein. Forexample, a nucleotide can lack a label moiety or a blocking moiety orboth. Examples of nucleotide analogues include, without limitation,7-deaza-adenine, 7-deaza-guanine, the analogues of deoxynucleotidesshown herein, analogues in which a label is attached through a cleavablelinker to the 5-position of cytosine or thymine or to the 7-position ofdeaza-adenine or deaza-guanine, and analogues in which a small chemicalmoiety is used to cap the OH group at the 3′-position of deoxyribose.Nucleotide analogues and DNA polymerase-based DNA sequencing are alsodescribed in U.S. Pat. Nos. 6,664,079, 10,738,072, and 11,174,281, eachwhich are incorporated herein by reference in their entirety for allpurposes.

As used herein, the term “associated” or “associated with” can mean thattwo or more species are identifiable as being co-located at a point intime. An association can mean that two or more species are or werewithin a similar container. An association can be an informaticsassociation, where for example digital information regarding two or morespecies is stored and can be used to determine that one or more of thespecies were co-located at a point in time. An association can also be aphysical association.

As used herein, the term “complementary” or “substantiallycomplementary” refers to the hybridization, base pairing, or theformation of a duplex between nucleotides or nucleic acids. For example,complementarity exists between the two strands of a double-stranded DNAmolecule or between an oligonucleotide primer and a primer binding siteon a single-stranded nucleic acid when a nucleotide (e.g., RNA or DNA)or a sequence of nucleotides is capable of base pairing with arespective cognate nucleotide or cognate sequence of nucleotides. Whenreferring to a double-stranded polynucleotide including a first strandhybridized to a second strand, it is to be understood that each of theterms “first strand” and “second strand” refer to single-strandedpolynucleotides. As described herein and commonly known in the art thecomplementary (matching) nucleotide of adenosine (A) is thymidine (T)and the complementary (matching) nucleotide of guanosine (G) is cytosine(C). Thus, a complement may include a sequence of nucleotides that basepair with corresponding complementary nucleotides of a second nucleicacid sequence. The nucleotides of a complement may partially orcompletely match the nucleotides of the second nucleic acid sequence.Where the nucleotides of the complement completely match each nucleotideof the second nucleic acid sequence, the complement forms base pairswith each nucleotide of the second nucleic acid sequence. Where thenucleotides of the complement partially match the nucleotides of thesecond nucleic acid sequence only some of the nucleotides of thecomplement form base pairs with nucleotides of the second nucleic acidsequence. Examples of complementary sequences include coding andnon-coding sequences, wherein the non-coding sequence containscomplementary nucleotides to the coding sequence and thus forms thecomplement of the coding sequence. A further example of complementarysequences are sense and antisense sequences, wherein the sense sequencecontains complementary nucleotides to the antisense sequence and thusforms the complement of the antisense sequence.

“Duplex” means at least two oligonucleotides and/or polynucleotides thatare fully or partially complementary undergo Watson-Crick type basepairing among all or most of their nucleotides so that a stable complexis formed. Complementary single stranded nucleic acids and/orsubstantially complementary single stranded nucleic acids can hybridizeto each other under hybridization conditions, thereby forming a nucleicacid that is partially or fully double stranded. When referring to adouble-stranded polynucleotide including a first strand hybridized to asecond strand, it is understood that each of the first strand and thesecond strand are independently single-stranded polynucleotides. All ora portion of a nucleic acid sequence may be substantially complementaryto another nucleic acid sequence, in some embodiments.

As referred to herein, “substantially complementary” refers tonucleotide sequences that can hybridize with each other under suitablehybridization conditions. Hybridization conditions can be altered totolerate varying amounts of sequence mismatch within complementarynucleic acids that are substantially complementary. Substantiallycomplementary portions of nucleic acids that can hybridize to each othercan be 75% or more, 76% or more, 77% or more, 78% or more, 79% or more,80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% ormore, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more or 99% or more complementary to eachother. In some embodiments substantially complementary portions ofnucleic acids that can hybridize to each other are 100% complementary.Nucleic acids, or portions thereof, that are configured to hybridize toeach other often comprise nucleic acid sequences that are substantiallycomplementary to each other.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,sequences in a pair of complementary sequences form portions of a singlepolynucleotide with non-base-pairing nucleotides (e.g., as in a hairpinor loop structure, with or without an overhang) or portions of separatepolynucleotides. In embodiments, one or both sequences in a pair ofcomplementary sequences form portions of longer polynucleotides, whichmay or may not include additional regions of complementarity.

As used herein, the term “contacting” is used in accordance with itsplain ordinary meaning and refers to the process of allowing at leasttwo distinct species (e.g. chemical compounds including biomolecules orcells) to become sufficiently proximal to react, interact or physicallytouch. However, the resulting reaction product can be produced directlyfrom a reaction between the added reagents or from an intermediate fromone or more of the added reagents that can be produced in the reactionmixture. The term “contacting” may include allowing two species toreact, interact, or physically touch, wherein the two species may be acompound, nucleic acid, a protein, or enzyme (e.g., a DNA polymerase).

As used herein, the terms “solid support” and “substrate” and “solidsurface” are used interchangeably and refers to discrete solid orsemi-solid surfaces to which a plurality of nucleic acid (e.g., primers)may be attached. A solid support may encompass any type of solid,porous, or hollow sphere, ball, cylinder, or other similar configurationcomposed of plastic, ceramic, metal, or polymeric material (e.g.,hydrogel) onto which a nucleic acid may be immobilized (e.g., covalentlyor non-covalently). A solid support may comprise a discrete particlethat may be spherical (e.g., microspheres) or have a non-spherical orirregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical,oblong, or disc-shaped, and the like. Solid supports may be in the formof discrete particles, which alone does not imply or require anyparticular shape. The term “particle” means a small body made of a rigidor semi-rigid material. The body can have a shape characterized, forexample, as a sphere, oval, microsphere, or other recognized particleshape whether having regular or irregular dimensions. As used herein,the term “discrete particles” refers to physically distinct particleshaving discernible boundaries. The term “particle” does not indicate anyparticular shape. The shapes and sizes of a collection of particles maybe different or about the same (e.g., within a desired range ofdimensions, or having a desired average or minimum dimension). Aparticle may be substantially spherical (e.g., microspheres) or have anon-spherical or irregular shape, such as cubic, cuboid, pyramidal,cylindrical, conical, oblong, or disc-shaped, and the like. Inembodiments, the particle has the shape of a sphere, cylinder,spherocylinder, or ellipsoid. Discrete particles collected in acontainer and contacting one another will define a bulk volumecontaining the particles, and will typically leave some internalfraction of that bulk volume unoccupied by the particles, even whenpacked closely together. In embodiments, cores and/or core-shellparticles are approximately spherical. As used herein the term“spherical” refers to structures which appear substantially or generallyof spherical shape to the human eye, and does not require a sphere to amathematical standard. In other words, “spherical” cores or particlesare generally spheroidal in the sense of resembling or approximating toa sphere. In embodiments, the diameter of a spherical core or particleis substantially uniform, e.g., about the same at any point, but maycontain imperfections, such as deviations of up to 1, 2, 3, 4, 5 or upto 10%. Because cores or particles may deviate from a perfect sphere,the term “diameter” refers to the longest dimension of a given core orparticle. Likewise, polymer shells are not necessarily of perfectuniform thickness all around a given core. Thus, the term “thickness” inrelation to a polymer structure (e.g., a shell polymer of a core-shellparticle) refers to the average thickness of the polymer layer.

A solid support may further comprise a polymer or hydrogel on thesurface to which the primers are attached (e.g., the primers arecovalently attached to the polymer, wherein the polymer is in directcontact with the solid support). Exemplary solid supports include, butare not limited to, glass and modified or functionalized glass, plastics(including acrylics, polystyrene and copolymers of styrene and othermaterials, polypropylene, polyethylene, polybutylene, polyurethanes,Teflon™, cyclic olefin copolymers, polyimides etc.), nylon, ceramics,resins, Zeonor, silica or silica-based materials including silicon andmodified silicon, carbon, metals, inorganic glasses, optical fiberbundles, photopatternable dry film resists, UV-cured adhesives andpolymers. The solid supports for some embodiments have at least onesurface located within a flow cell. The solid support, or regionsthereof, can be substantially flat. The solid support can have surfacefeatures such as wells, pits, channels, ridges, raised regions, pegs,posts or the like. The term solid support is encompassing of a substrate(e.g., a flow cell) having a surface comprising a polymer coatingcovalently attached thereto. In embodiments, the solid support is a flowcell. The term “flow cell” as used herein refers to a chamber includinga solid surface across which one or more fluid reagents can be flowed.Examples of flow cells and related fluidic systems and detectionplatforms that can be readily used in the methods of the presentdisclosure are described, for example, in Bentley et al., Nature456:53-59 (2008). In certain embodiments a substrate comprises a surface(e.g., a surface of a flow cell, a surface of a tube, a surface of achip, surface of a particle), for example a metal surface (e.g., steel,gold, silver, aluminum, silicon and copper). In some embodiments asubstrate (e.g., a substrate surface) is coated and/or comprisesfunctional groups and/or inert materials. In certain embodiments asubstrate comprises a bead, a chip, a capillary, a plate, a membrane, awafer (e.g., silicon wafers), a comb, or a pin for example. In someembodiments a substrate comprises a bead and/or a nanoparticle. Asubstrate can be made of a suitable material, non-limiting examples ofwhich include a plastic or a suitable polymer (e.g., polycarbonate,poly(vinyl alcohol), poly(divinylbenzene), polystyrene, polyamide,polyester, polyvinylidene difluoride (PVDF), polyethylene, polyurethane,polypropylene, and the like), borosilicate, silica, nylon, Wang resin,Merrifield resin, metal (e.g., iron, a metal alloy, sepharose, agarose,polyacrylamide, dextran, cellulose and the like or combinations thereof.In some embodiments a substrate comprises a magnetic material (e.g.,iron, nickel, cobalt, platinum, aluminum, and the like). In certainembodiments a substrate comprises a magnetic bead (e.g., DYNABEADS®,hematite, AMPure XP). Magnets can be used to purify and/or capturenucleic acids bound to certain substrates (e.g., substrates comprising ametal or magnetic material).

As used herein, the term “polymer” refers to macromolecules having oneor more structurally unique repeating units. The repeating units arereferred to as “monomers,” which are polymerized for the polymer.Typically, a polymer is formed by monomers linked in a chain-likestructure. A polymer formed entirely from a single type of monomer isreferred to as a “homopolymer.” A polymer formed from two or more uniquerepeating structural units may be referred to as a “copolymer.” Apolymer may be linear or branched, and may be random, block, polymerbrush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, orpolymer micelles. The term “polymer” includes homopolymers, copolymers,tripolymers, tetra polymers and other polymeric molecules made frommonomeric subunits. Copolymers include alternating copolymers, periodiccopolymers, statistical copolymers, random copolymers, block copolymers,linear copolymers and branched copolymers. The term “polymerizablemonomer” is used in accordance with its meaning in the art of polymerchemistry and refers to a compound that may covalently bind chemicallyto other monomer molecules (such as other polymerizable monomers thatare the same or different) to form a polymer. Polymers can behydrophilic, hydrophobic, or amphiphilic, as known in the art. Thus,“hydrophilic polymers” are substantially miscible with water andinclude, but are not limited to, polyethylene glycol and the like.“Hydrophobic polymers” are substantially immiscible with water andinclude, but are not limited to, polyethylene, polypropylene,polybutadiene, polystyrene, polymers disclosed herein, and the like.“Amphiphilic polymers” have both hydrophilic and hydrophobic propertiesand are typically copolymers having hydrophilic segment(s) andhydrophobic segment(s). Polymers include homopolymers, randomcopolymers, and block copolymers, as known in the art. The term“homopolymer” refers, in the usual and customary sense, to a polymerhaving a single monomeric unit. The term “copolymer” refers to a polymerderived from two or more monomeric species. The term “random copolymer”refers to a polymer derived from two or more monomeric species with nopreferred ordering of the monomeric species. The term “block copolymer”refers to polymers having two or homopolymer subunits linked by covalentbond. Thus, the term “hydrophobic homopolymer” refers to a homopolymerwhich is hydrophobic. The term “hydrophobic block copolymer” refers totwo or more homopolymer subunits linked by covalent bonds and which ishydrophobic.

As used herein, the term “hydrogel” refers to a three-dimensionalpolymeric structure that is substantially insoluble in water, but whichis capable of absorbing and retaining large quantities of water to forma substantially stable, often soft and pliable, structure. Inembodiments, water can penetrate in between polymer chains of a polymernetwork, subsequently causing swelling and the formation of a hydrogel.In embodiments, hydrogels are super-absorbent (e.g., containing morethan about 90% water) and can be comprised of natural or syntheticpolymers.

The term “surface” is intended to mean an external part or externallayer of a substrate. The surface can be in contact with anothermaterial such as a gas, liquid, gel, polymer, organic polymer, secondsurface of a similar or different material, metal, or coating. Thesurface, or regions thereof, can be substantially flat. The substrateand/or the surface can have surface features such as wells, pits,channels, ridges, raised regions, pegs, posts or the like.

As used herein, the terms “cluster” and “colony” are usedinterchangeably to refer to a discrete site on a solid support thatincludes a plurality of immobilized polynucleotides and a plurality ofimmobilized complementary polynucleotides. The term “clustered array”refers to an array formed from such clusters or colonies. In thiscontext the term “array” is not to be understood as requiring an orderedarrangement of clusters. The term “array” is used in accordance with itsordinary meaning in the art, and refers to a population of differentmolecules that are attached to one or more solid-phase substrates suchthat the different molecules can be differentiated from each otheraccording to their relative location. An array can include differentmolecules that are each located at different addressable features on asolid-phase substrate. The molecules of the array can be nucleic acidprimers, nucleic acid probes, nucleic acid templates or nucleic acidenzymes such as polymerases or ligases. Arrays useful in the inventioncan have densities that ranges from about 2 different features to manymillions, billions or higher. The density of an array can be from 2 toas many as a billion or more different features per square cm. Forexample an array can have at least about 100 features/cm², at leastabout 1,000 features/cm², at least about 10,000 features/cm², at leastabout 100,000 features/cm², at least about 10,000,000 features/cm², atleast about 100,000,000 features/cm², at least about 1,000,000,000features/cm², at least about 2,000,000,000 features/cm² or higher. Inembodiments, the arrays have features at any of a variety of densitiesincluding, for example, at least about 10 features/cm², 100features/cm², 500 features/cm², 1,000 features/cm², 5,000 features/cm²,10,000 features/cm², 50,000 features/cm², 100,000 features/cm²,1,000,000 features/cm², 5,000,000 features/cm², or higher.

As used herein, the term “rolling circle amplification (RCA)” refers toa nucleic acid amplification reaction that amplifies a circular nucleicacid template (e.g., single-stranded DNA circles) via a rolling circlemechanism. Rolling circle amplification reaction is initiated by thehybridization of a primer to a circular, often single-stranded, nucleicacid template. The nucleic acid polymerase then extends the primer thatis hybridized to the circular nucleic acid template by continuouslyprogressing around the circular nucleic acid template to replicate thesequence of the nucleic acid template over and over again (rollingcircle mechanism). The rolling circle amplification typically producesconcatemers comprising tandem repeat units of the circular nucleic acidtemplate sequence. The rolling circle amplification may be a linear RCA(LRCA), exhibiting linear amplification kinetics (e.g., RCA using asingle specific primer), or may be an exponential RCA (ERCA) exhibitingexponential amplification kinetics. Rolling circle amplification mayalso be performed using multiple primers (multiply primed rolling circleamplification or MPRCA) leading to hyper-branched concatemers. Forexample, in a double-primed RCA, one primer may be complementary, as inthe linear RCA, to the circular nucleic acid template, whereas the othermay be complementary to the tandem repeat unit nucleic acid sequences ofthe RCA product. Consequently, the double-primed RCA may proceed as achain reaction with exponential (geometric) amplification kineticsfeaturing a ramifying cascade of multiple-hybridization,primer-extension, and strand-displacement events involving both theprimers. This often generates a discrete set of concatemeric,double-stranded nucleic acid amplification products. The rolling circleamplification may be performed in-vitro under isothermal conditionsusing a suitable nucleic acid polymerase such as a polymerase describedherein, including embodiments. RCA may be performed by using any of theDNA polymerases that are known in the art (e.g., a Phi29 DNA polymerase,a Bst DNA polymerase, or SD polymerase).

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically and preferably conducted with oligonucleotides. The terms“annealing” and “hybridization” are used interchangeably to mean theformation of a stable duplex. In some embodiments, one portion of anucleic acid hybridizes to itself, such as in the formation of a hairpinstructure. The propensity for hybridization between nucleic acidsdepends on the temperature and ionic strength of their milieu, thelength of the nucleic acids and the degree of complementarity. Theeffect of these parameters on hybridization is described in, forexample, Sambrook J., Fritsch E. F., Maniatis T., Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratory Press, New York (1989).As used herein, hybridization of a primer, or of a DNA extensionproduct, respectively, is extendable by creation of a phosphodiesterbond with an available nucleotide or nucleotide analogue capable offorming a phosphodiester bond, therewith. For example, hybridization canbe performed at a temperature ranging from 15° C. to 95° C. In someembodiments, the hybridization is performed at a temperature of about20° C., about 25° C., about 30° C., about 35° C., about 40° C., about45° C., about 50° C., about 55° C., about 60° C., about 65° C., about70° C., about 75° C., about 80° C., about 85° C., about 90° C., or about95° C. In other embodiments, the stringency of the hybridization can befurther altered by the addition or removal of components of the bufferedsolution. Those skilled in the art understand how to estimate and adjustthe stringency of hybridization conditions such that sequences having atleast a desired level of complementarity will stably hybridize, whilethose having lower complementarity will not. As used herein, the term“stringent condition” refers to condition(s) under which apolynucleotide probe or primer will hybridize preferentially to itstarget sequence, and to a lesser extent to, or not at all to, othersequences. In some embodiments nucleic acids, or portions thereof, thatare configured to specifically hybridize are often about 80% or more,81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, 99% or more or 100% complementary to each other overa contiguous portion of nucleic acid sequence. A specific hybridizationdiscriminates over non-specific hybridization interactions (e.g., twonucleic acids that a not configured to specifically hybridize, e.g., twonucleic acids that are 80% or less, 70% or less, 60% or less or 50% orless complementary) by about 2-fold or more, often about 10-fold ormore, and sometimes about 100-fold or more, 1000-fold or more,10,000-fold or more, 100,000-fold or more, or 1,000,000-fold or more.Two nucleic acid strands (e.g., two single-stranded polynucleotides)that are hybridized to each other can form a duplex which comprises adouble-stranded portion of nucleic acid.

II. Compositions & Kits

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including at least one mutation at amino acidposition 7 or an amino acid position corresponding to position 7,wherein the mutation at amino acid position 7 includes histidine,lysine, or arginine; amino acid position 97 or an amino acid positioncorresponding to position 97, wherein the mutation at amino acidposition 97 includes cysteine, histidine, lysine, serine, threonine, ormethionine; amino acid position 579 or an amino acid positioncorresponding to position 579, wherein the mutation at amino acidposition 579 includes leucine, isoleucine, valine, alanine, or glycine;amino acid position 588 or an amino acid position corresponding toposition 588, wherein the mutation at amino acid position 588 includesleucine, isoleucine, valine, alanine, or glycine; or amino acid position742 or an amino acid position corresponding to position 742, wherein themutation at amino acid position 742 includes leucine, isoleucine,alanine, or glycine.

In another aspect is provided a polymerase including an amino acidsequence that is at least 80% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1; including a first mutation at amino acidposition 97 or an amino acid position corresponding to position 97,wherein the first mutation includes cysteine, histidine, lysine, serine,threonine, or methionine; a second mutation at amino acid position 588or an amino acid position corresponding to position 588; wherein thesecond mutation includes leucine, isoleucine, valine, alanine, orglycine.

In embodiments, the first mutation is cysteine, histidine, or serine. Inembodiments, the first mutation is cysteine. In embodiments, the firstmutation is histidine. In embodiments, the first mutation is serine.

In embodiments, the second mutation is leucine, isoleucine, valine, oralanine. In embodiments, the second mutation is leucine. In embodiments,the second mutation is isoleucine. In embodiments, the second mutationis alanine. In embodiments, the second mutation is valine.

In embodiments, the polymerase further includes a mutation at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is leucine, isoleucine, alanine, or glycine. Inembodiments, the polymerase further includes a mutation at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is leucine. In embodiments, the polymerase furtherincludes a mutation at amino acid position 742 or an amino acid positioncorresponding to position 742, wherein the mutation is isoleucine. Inembodiments, the polymerase further includes a mutation at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is alanine, or glycine. In embodiments, thepolymerase further includes a mutation at amino acid position 742 or anamino acid position corresponding to position 742, wherein the mutationis alanine. In embodiments, the polymerase further includes a mutationat amino acid position 742 or an amino acid position corresponding toposition 742, wherein the mutation is glycine.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including a first mutation at amino acid position588 or an amino acid position corresponding to position 588; wherein thefirst mutation is leucine, isoleucine, valine, alanine, or glycine; anda second mutation at amino acid position 7 or an amino acid positioncorresponding to position 7, wherein the second mutation is histidine,lysine, or arginine.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including a first mutation at amino acid position588 or an amino acid position corresponding to position 588; wherein thefirst mutation is leucine, isoleucine, valine, alanine, or glycine; anda second mutation at amino acid position 97 or an amino acid positioncorresponding to position 97, wherein the second mutation is cysteine,histidine, lysine, serine, threonine, or methionine.

In an aspect is provided a polymerase including an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; including a first mutation at amino acid position588 or an amino acid position corresponding to position 588; wherein thefirst mutation is leucine, isoleucine, valine, alanine, or glycine; anda second mutation at amino acid position 742 or an amino acid positioncorresponding to position 742, wherein the second mutation is leucine,isoleucine, alanine, or glycine.

In embodiments, the polymerase further includes a mutation at amino acidposition 726 or an amino acid position corresponding to amino acidposition 726, wherein the mutation is aspartic acid, glutamic acid,asparagine, or glutamine. In embodiments, the polymerase furtherincludes a mutation at amino acid position 726 or an amino acid positioncorresponding to amino acid position 726, wherein the mutation isaspartic acid. In embodiments, the polymerase further includes amutation at amino acid position 726 or an amino acid positioncorresponding to amino acid position 726, wherein the mutation isglutamic acid. In embodiments, the polymerase further includes amutation at amino acid position 726 or an amino acid positioncorresponding to amino acid position 726, wherein the mutation isasparagine. In embodiments, the polymerase further includes a mutationat amino acid position 726 or an amino acid position corresponding toamino acid position 726, wherein the mutation is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 769 or an amino acid position corresponding to position 769,wherein the mutation is leucine, isoleucine, arginine, valine, alanine,or glycine. In embodiments, the polymerase includes a mutation at aminoacid position 769 or an amino acid position corresponding to position769, wherein the mutation is leucine. In embodiments, the polymeraseincludes a mutation at amino acid position 769 or an amino acid positioncorresponding to position 769, wherein the mutation is isoleucine. Inembodiments, the polymerase includes a mutation at amino acid position769 or an amino acid position corresponding to position 769, wherein themutation is valine. In embodiments, the polymerase includes a mutationat amino acid position 769 or an amino acid position corresponding toposition 769, wherein the mutation is alanine. In embodiments, thepolymerase includes a mutation at amino acid position 769 or an aminoacid position corresponding to position 769, wherein the mutation isglycine. In embodiments, the polymerase includes a mutation at aminoacid position 769 or an amino acid position corresponding to position769, wherein the mutation is arginine.

In embodiments, the polymerase includes a mutation at amino acidposition 7 or an amino acid position corresponding to position 7,wherein the mutation is histidine, lysine, or arginine. In embodiments,the polymerase includes a mutation at amino acid position 7 or an aminoacid position corresponding to position 7, wherein the mutation ishistidine. In embodiments, the polymerase includes a mutation at aminoacid position 7 or an amino acid position corresponding to position 7,wherein the mutation is lysine. In embodiments, the polymerase includesa mutation at amino acid position 7 or an amino acid positioncorresponding to position 7, wherein the mutation is arginine.

In embodiments, the polymerase includes a mutation at amino acidposition 13 or an amino acid position corresponding to position 13,wherein the mutation is arginine, isoleucine, methionine, or histidine.In embodiments, the polymerase includes a mutation at amino acidposition 13 or an amino acid position corresponding to position 13,wherein the mutation is arginine or histidine. In embodiments, thepolymerase includes a mutation at amino acid position 13 or an aminoacid position corresponding to position 13, wherein the mutation isarginine. In embodiments, the polymerase includes a mutation at aminoacid position 13 or an amino acid position corresponding to position 13,wherein the mutation is histidine. In embodiments, the polymeraseincludes a mutation at amino acid position 13 or an amino acid positioncorresponding to position 13, wherein the mutation is methionine. Inembodiments, the polymerase includes a mutation at amino acid position13 or an amino acid position corresponding to position 13, wherein themutation is isoleucine. In embodiments, the polymerase includes amutation at amino acid position 13 or an amino acid positioncorresponding to position 13, wherein the mutation is arginine, leucine,isoleucine, or histidine.

In embodiments, the polymerase includes a mutation at amino acidposition 13 or an amino acid position corresponding to position 13,wherein the mutation is arginine, leucine, isoleucine, or histidine; anda mutation at amino acid position 579 or an amino acid positioncorresponding to position 579, wherein the mutation is alanine orglycine.

In embodiments, the polymerase includes a mutation at amino acidposition 40 or an amino acid position corresponding to position 40,wherein the mutation is valine, leucine, or threonine. In embodiments,the mutation at amino acid position 40 is valine. In embodiments, themutation at amino acid position 40 is leucine. In embodiments, themutation at amino acid position 40 is threonine.

In embodiments, the polymerase includes a mutation at amino acidposition 75 or an amino acid position corresponding to position 75,wherein the mutation at amino acid position 75 is cysteine, histidine,lysine, serine, threonine, or methionine. In embodiments, the mutationat amino acid position 75 is cysteine. In embodiments, the mutation atamino acid position 75 is histidine. In embodiments, the mutation atamino acid position 75 is lysine. In embodiments, the mutation at aminoacid position 75 is serine. In embodiments, the mutation at amino acidposition 75 is threonine. In embodiments, the mutation at amino acidposition 75 is methionine.

In embodiments, the polymerase includes a glutamine, valine, arginine,or alanine at amino acid position 93 or an amino acid positioncorresponding to position 93. In embodiments, the mutation at amino acidposition 93 is glutamine, valine, arginine, or alanine. In embodiments,the mutation at amino acid position 93 is glutamine. In embodiments, themutation at amino acid position 93 is valine. In embodiments, themutation at amino acid position 93 is arginine. In embodiments, themutation at amino acid position 93 is alanine. In embodiments, thepolymerase includes a glutamine, valine, arginine, or alanine at aminoacid position 93 or the amino acid position corresponding to position93. In embodiments, the polymerase includes a glutamine, valine,arginine, or alanine at amino acid position 93. It is known that thepresence of uracil in DNA results in a dramatic increase in the bindingaffinity of archaeal family B DNA polymerases, stalling furtherpolymerase activity (Lasken R S et al. J. Biol. Chem. 1996, 271(30):17692-6 and Fogg M J et al. Nature Structural Biology. 2002, 9:922-7). A specific point mutation in the uracil-binding pocket of thesepolymerases disrupts uracil binding and allows extension in the presenceof uracil without compromising polymerase activity (Norholm MH BMCBiotechnology. 2010, 10:21). Provided herein are novel DNA polymerasevariants (e.g., V93Q, V93R, V93A) that disrupt the uracil bindingpocket. In embodiments, the polymerase includes a V93Q, V93R, or V93Amutation. In embodiments, the polymerase includes a V93Q mutation. Inembodiments, the polymerase includes a V931, V93L, V93N, V93D, or V93Emutation. In embodiments, the polymerase includes an amino acidsubstitution at position 93. In embodiments, the amino acid substitutionat position 93 is a glutamine substitution. In embodiments, the aminoacid substitution at position 93 is an arginine substitution. Inembodiments, the amino acid substitution at position 93 is an alaninesubstitution. In embodiments, the amino acid substitution at position 93is a leucine substitution. In embodiments, the amino acid substitutionat position 93 is an isoleucine substitution.

In embodiments, the polymerase includes a mutation at amino acidposition 97 or an amino acid position corresponding to position 97,wherein the mutation at amino acid position 97 is cysteine, histidine,leucine, lysine, serine, threonine, or methionine. In embodiments, themutation at amino acid position 97 is cysteine. In embodiments, themutation at amino acid position 97 is histidine. In embodiments, themutation at amino acid position 97 is lysine. In embodiments, themutation at amino acid position 97 is serine. In embodiments, themutation at amino acid position 97 is threonine. In embodiments, themutation at amino acid position 97 is methionine. In embodiments, themutation at amino acid position 97 is leucine.

In embodiments, the polymerase includes a mutation at amino acidposition 160 or an amino acid position corresponding to position 160,wherein the mutation is leucine, valine, alanine, or glycine. Inembodiments, the mutation at amino acid position 160 is leucine. Inembodiments, the mutation at amino acid position 160 is valine. Inembodiments, the mutation at amino acid position 160 is alanine. Inembodiments, the mutation at amino acid position 160 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 168 or an amino acid position corresponding to position 168,wherein the mutation is threonine, serine, cysteine, or glycine. Inembodiments, the mutation at amino acid position 168 is threonine. Inembodiments, the mutation at amino acid position 168 is serine. Inembodiments, the mutation at amino acid position 168 is cysteine. Inembodiments, the mutation at amino acid position 168 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 214 or an amino acid position corresponding to position 214,wherein the mutation is leucine, alanine, isoleucine, serine, threonine,or glycine. In embodiments, the mutation at amino acid position 214 isleucine. In embodiments, the mutation at amino acid position 214 isalanine. In embodiments, the mutation at amino acid position 214 isisoleucine. In embodiments, the mutation at amino acid position 214 isglycine. In embodiments, the mutation at amino acid position 214 isthreonine. In embodiments, the mutation at amino acid position 214 isserine.

In embodiments, the polymerase includes a mutation at amino acidposition 241 or an amino acid position corresponding to position 241,wherein the mutation is leucine, isoleucine, alanine, valine, orglycine. In embodiments, the polymerase includes a mutation at aminoacid position 241 or an amino acid position corresponding to position241, wherein the mutation is leucine. In embodiments, the polymeraseincludes a mutation at amino acid position 241 or an amino acid positioncorresponding to position 241, wherein the mutation is isoleucine. Inembodiments, the polymerase includes a mutation at amino acid position241 or an amino acid position corresponding to position 241, wherein themutation is alanine. In embodiments, the polymerase includes a mutationat amino acid position 241 or an amino acid position corresponding toposition 241, wherein the mutation is valine. In embodiments, thepolymerase includes a mutation at amino acid position 241 or an aminoacid position corresponding to position 241, wherein the mutation isglycine.

In embodiments, the polymerase includes a mutation at amino acidposition 281 or an amino acid position corresponding to position 281,wherein the mutation is threonine, serine, cysteine, or glycine. Inembodiments, the mutation at amino acid position 281 is threonine. Inembodiments, the mutation at amino acid position 281 is serine. Inembodiments, the mutation at amino acid position 281 is cysteine. Inembodiments, the mutation at amino acid position 281 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 292 or an amino acid position corresponding to position 292,wherein the mutation is glutamic acid, valine, leucine, or glycine. Inembodiments, the mutation at amino acid position 292 is glutamic acid.In embodiments, the mutation at amino acid position 292 is valine. Inembodiments, the mutation at amino acid position 292 is leucine. Inembodiments, the mutation at amino acid position 292 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 316 or an amino acid position corresponding to position 316,wherein the mutation is proline or glycine. In embodiments, the mutationat amino acid position 316 is proline. In embodiments, the mutation atamino acid position 316 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 350 or an amino acid position corresponding to position 350,wherein the mutation is threonine or serine. In embodiments, themutation at amino acid position 350 is serine. In embodiments, themutation at amino acid position 350 is threonine.

In embodiments, the polymerase includes a mutation at amino acidposition 465 or an amino acid position corresponding to position 465,wherein the mutation is asparagine, aspartic acid, glutamic acid,threonine, or glutamine. In embodiments, the polymerase includes amutation at amino acid position 465 or an amino acid positioncorresponding to position 465, wherein the mutation is asparagine. Inembodiments, the polymerase includes a mutation at amino acid position465 or an amino acid position corresponding to position 465, wherein themutation is aspartic acid. In embodiments, the polymerase includes amutation at amino acid position 465 or an amino acid positioncorresponding to position 465, wherein the mutation is glutamic acid. Inembodiments, the polymerase includes a mutation at amino acid position465 or an amino acid position corresponding to position 465, wherein themutation is glutamine. In embodiments, the polymerase includes amutation at amino acid position 465 or an amino acid positioncorresponding to position 465, wherein the mutation is threonine.

In embodiments, the polymerase includes a mutation at amino acidposition 469 or an amino acid position corresponding to position 469,wherein the mutation is threonine, serine, cysteine, or methionine. Inembodiments, the polymerase includes a mutation at amino acid position469 or an amino acid position corresponding to position 469, wherein themutation is threonine. In embodiments, the polymerase includes amutation at amino acid position 469 or an amino acid positioncorresponding to position 469, wherein the mutation is serine. Inembodiments, the polymerase includes a mutation at amino acid position469 or an amino acid position corresponding to position 469, wherein themutation is cysteine. In embodiments, the polymerase includes a mutationat amino acid position 469 or an amino acid position corresponding toposition 469, wherein the mutation is methionine.

In embodiments, the polymerase includes a mutation at amino acidposition 469 or an amino acid position corresponding to position 469,wherein the mutation is asparagine, aspartic acid, glutamic acid, orglutamine. In embodiments, the polymerase includes a mutation at aminoacid position 469 or an amino acid position corresponding to position469, wherein the mutation is asparagine. In embodiments, the polymeraseincludes a mutation at amino acid position 469 or an amino acid positioncorresponding to position 469, wherein the mutation is aspartic acid. Inembodiments, the polymerase includes a mutation at amino acid position469 or an amino acid position corresponding to position 469, wherein themutation is glutamic acid. In embodiments, the polymerase includes amutation at amino acid position 469 or an amino acid positioncorresponding to position 469, wherein the mutation is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 472 or an amino acid position corresponding to position 472,wherein the mutation is asparagine, aspartic acid, glutamic acid, orglutamine. In embodiments, the polymerase includes a mutation at aminoacid position 472 or an amino acid position corresponding to position472, wherein the mutation is asparagine. In embodiments, the polymeraseincludes a mutation at amino acid position 472 or an amino acid positioncorresponding to position 472, wherein the mutation is aspartic acid. Inembodiments, the polymerase includes a mutation at amino acid position472 or an amino acid position corresponding to position 472, wherein themutation is glutamic acid. In embodiments, the polymerase includes amutation at amino acid position 472 or an amino acid positioncorresponding to position 472, wherein the mutation is glutamine. Inembodiments, the polymerase includes a mutation at amino acid position472 or an amino acid position corresponding to position 472, wherein themutation is asparagine or glutamic acid.

In embodiments, the polymerase includes a mutation at amino acidposition 477 or an amino acid position corresponding to position 477,wherein the mutation is isoleucine, leucine, tryptophan, phenylalanine,alanine, or glycine. In embodiments, the mutation at amino acid position477 is isoleucine. In embodiments, the mutation at amino acid position477 is leucine. In embodiments, the mutation at amino acid position 477is alanine. In embodiments, the mutation at amino acid position 477 isglycine. In embodiments, the mutation at amino acid position 477 istryptophan. In embodiments, the mutation at amino acid position 477 isphenylalanine.

In embodiments, the polymerase includes a mutation at amino acidposition 491 or an amino acid position corresponding to position 491,wherein the mutation is glycine, valine, leucine, or isoleucine. Inembodiments, the polymerase includes a mutation at amino acid position491 or an amino acid position corresponding to position 491, wherein themutation is glycine. In embodiments, the polymerase includes a mutationat amino acid position 491 or an amino acid position corresponding toposition 491, wherein the mutation is valine. In embodiments, thepolymerase includes a mutation at amino acid position 491 or an aminoacid position corresponding to position 491, wherein the mutation isleucine. In embodiments, the polymerase includes a mutation at aminoacid position 491 or an amino acid position corresponding to position491, wherein the mutation is isoleucine.

In embodiments, the polymerase includes a mutation at amino acidposition 520 or an amino acid position corresponding to position 520,wherein the mutation is histidine, lysine, or arginine. In embodiments,the polymerase includes a mutation at amino acid position 520 or anamino acid position corresponding to position 520, wherein the mutationis histidine. In embodiments, the polymerase includes a mutation atamino acid position 520 or an amino acid position corresponding toposition 520, wherein the mutation is lysine. In embodiments, thepolymerase includes a mutation at amino acid position 520 or an aminoacid position corresponding to position 520, wherein the mutation isarginine.

In embodiments, the polymerase includes a mutation at amino acidposition 526 or an amino acid position corresponding to position 526,wherein the mutation is histidine, lysine, or glutamine. In embodiments,the mutation at amino acid position 526 is histidine. In embodiments,the mutation at amino acid position 526 is lysine. In embodiments, themutation at amino acid position 526 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 579 or an amino acid position corresponding to position 579,wherein the mutation is alanine or glycine. In embodiments, the mutationat amino acid position 579 is alanine. In embodiments, the mutation atamino acid position 579 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 563 or an amino acid position corresponding to amino acidposition 563, wherein the mutation is aspartic acid, glycine,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 563 is aspartic acid. In embodiments, the mutation at aminoacid position 563 is glycine. In embodiments, the mutation at amino acidposition 563 is asparagine. In embodiments, the mutation at amino acidposition 563 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 577 or an amino acid position corresponding to amino acidposition 577, wherein the mutation is aspartic acid, lysine, glycine,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 577 is aspartic acid. In embodiments, the mutation at aminoacid position 577 is glycine. In embodiments, the mutation at amino acidposition 577 is asparagine. In embodiments, the mutation at amino acidposition 577 is glutamine. In embodiments, the mutation at amino acidposition 577 is lysine.

In embodiments, the polymerase includes a mutation at amino acidposition 579 or an amino acid position corresponding to amino acidposition 579, wherein the mutation is aspartic acid, lysine, glycine,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 577 is aspartic acid. In embodiments, the mutation at aminoacid position 577 is lysine. In embodiments, the mutation at amino acidposition 577 is glycine. In embodiments, the mutation at amino acidposition 577 is asparagine. In embodiments, the mutation at amino acidposition 577 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 588 or an amino acid position corresponding to position 588;wherein the second mutation is leucine, isoleucine, valine, alanine, orglycine. In embodiments, the mutation at amino acid position 588 or anamino acid position corresponding to position 588 is leucine. Inembodiments, the mutation at amino acid position 588 or an amino acidposition corresponding to position 588 is isoleucine. In embodiments,the mutation at amino acid position 588 or an amino acid positioncorresponding to position 588 is valine. In embodiments, the mutation atamino acid position 588 or an amino acid position corresponding toposition 588 is alanine. In embodiments, the mutation at amino acidposition 588 or an amino acid position corresponding to position 588 isglycine. In embodiments, the mutation at amino acid position 588 or anamino acid position corresponding to position 588 is leucine,isoleucine, valine, or alanine. In embodiments, the mutation at aminoacid position 588 or an amino acid position corresponding to position588 is leucine or valine.

In embodiments, the polymerase includes a mutation at amino acidposition 601 or an amino acid position corresponding to amino acidposition 601, wherein the mutation is aspartic acid, lysine, glycine,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 601 is aspartic acid. In embodiments, the mutation at aminoacid position 601 is lysine. In embodiments, the mutation at amino acidposition 601 is glycine. In embodiments, the mutation at amino acidposition 601 is asparagine. In embodiments, the mutation at amino acidposition 601 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 635 or an amino acid position corresponding to amino acidposition 635, wherein the mutation is aspartic acid, lysine, glutamicacid, asparagine, or glutamine. In embodiments, the mutation at aminoacid position 635 is aspartic acid. In embodiments, the mutation atamino acid position 635 is glutamic acid. In embodiments, the mutationat amino acid position 635 is asparagine. In embodiments, the mutationat amino acid position 635 is glutamine. In embodiments, the mutation atamino acid position 635 is lysine.

In embodiments, the polymerase includes a mutation at amino acidposition 637 or an amino acid position corresponding to amino acidposition 637, wherein the mutation is aspartic acid, lysine, glutamicacid, asparagine, or glutamine. In embodiments, the mutation at aminoacid position 637 is aspartic acid. In embodiments, the mutation atamino acid position 637 is glutamic acid. In embodiments, the mutationat amino acid position 637 is asparagine. In embodiments, the mutationat amino acid position 637 is glutamine. In embodiments, the mutation atamino acid position 637 is lysine.

In embodiments, the polymerase includes a mutation at amino acidposition 655 or an amino acid position corresponding to amino acidposition 655, wherein the mutation is aspartic acid, lysine, glycine,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 655 is aspartic acid. In embodiments, the mutation at aminoacid position 655 is lysine. In embodiments, the mutation at amino acidposition 655 is glycine. In embodiments, the mutation at amino acidposition 655 is asparagine. In embodiments, the mutation at amino acidposition 655 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 706 or an amino acid position corresponding to position 706,wherein the mutation at amino acid position 706 is cysteine, histidine,leucine, lysine, serine, threonine, or methionine. In embodiments, themutation at amino acid position 706 is cysteine. In embodiments, themutation at amino acid position 706 is histidine. In embodiments, themutation at amino acid position 706 is lysine. In embodiments, themutation at amino acid position 706 is serine. In embodiments, themutation at amino acid position 706 is threonine. In embodiments, themutation at amino acid position 706 is methionine. In embodiments, themutation at amino acid position 706 is leucine.

In embodiments, the polymerase includes a mutation at amino acidposition 726 or an amino acid position corresponding to amino acidposition 726, wherein the mutation is aspartic acid, glutamic acid,asparagine, or glutamine. In embodiments, the mutation at amino acidposition 726 is aspartic acid. In embodiments, the mutation at aminoacid position 726 is glutamic acid. In embodiments, the mutation atamino acid position 726 is asparagine. In embodiments, the mutation atamino acid position 726 is glutamine.

In embodiments, the polymerase includes a mutation at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is leucine, isoleucine, alanine, or glycine. Inembodiments, the mutation at amino acid position 742 is leucine. Inembodiments, the mutation at amino acid position 742 is isoleucine. Inembodiments, the mutation at amino acid position 742 is alanine. Inembodiments, the mutation at amino acid position 742 is glycine.

In embodiments, the polymerase includes a mutation at amino acidposition 762 or an amino acid position corresponding to position 762,wherein the mutation is asparagine, glutamine, threonine, or serine. Inembodiments, the mutation at amino acid position 762 is asparagine. Inembodiments, the mutation at amino acid position 762 is glutamine. Inembodiments, the mutation at amino acid position 762 is threonine. Inembodiments, the mutation at amino acid position 762 is serine.

In embodiments, the polymerase includes an alanine at amino acidposition 141 or an amino acid position corresponding to position 141;and an alanine at amino acid position 143 or an amino acid positioncorresponding to position 143. In embodiments, the polymerase includesan alanine at amino acid position 141 or the amino acid positioncorresponding to position 141; and an alanine at amino acid position 143or the amino acid position corresponding to position 143. Inembodiments, the polymerase includes an alanine at amino acid position141; and an alanine at amino acid position 143.

In embodiments, the polymerase includes an amino acid substitution atposition 141. In embodiments, the amino acid substitution at position141 is an alanine substitution. In embodiments, the amino acidsubstitution at position 141 is a glycine substitution.

In embodiments, the polymerase includes an amino acid substitution atposition 143. In embodiments, the amino acid substitution at position143 is an alanine substitution. In embodiments, the amino acidsubstitution at position 143 is a glycine, alanine, threonine, or serinesubstitution.

In embodiments, the polymerase does not include a mutation at amino acidpositions 409 and/or 410.

In embodiments, the polymerase includes, relative to SEQ ID NO:1, R97H,F588L, G635D, and V742A. In embodiments, the polymerase includes,relative to SEQ ID NO:1, K13R, R97C, E579G, and F588L. In embodiments,the polymerase includes, relative to SEQ ID NO:1. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, R97C, E563G, E579G, F588L,and V742A. In embodiments, the polymerase includes, relative to SEQ IDNO:1, R97H, F588L, and G635D. In embodiments, the polymerase includes,relative to SEQ ID NO:1, R97C, F588L, and V742A. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, Y7H, K13I, R97L, E579G,F588L, and V742A. In embodiments, the polymerase includes, relative toSEQ ID NO:1, Y7H, K13R, R97H, and V742A. In embodiments, the polymeraseincludes, relative to SEQ ID NO:1, Y7H, K13R, R97C, D141A, E579G, F588L,and V742A. In embodiments, the polymerase includes, relative to SEQ IDNO:1, R97C, E579G, F588L, and V742A. In embodiments, the polymeraseincludes, relative to SEQ ID NO:1, Y7H, R97C, E579G, and F588L. Inembodiments, the polymerase includes, relative to SEQ ID NO:1, Y7H,R97C, and V742A. In embodiments, the polymerase includes, relative toSEQ ID NO:1, Y7H, K13R, E563G, F588L, and V742A. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, E29D, Y30F, R97H, I160V,K229E, A511V, I548V, F588L, G635D, and V742A. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, K13R, R32H, F75L, R97C,K201I, Y209H, I256V, Y291H, E383D, G400D, R526C, E579G, F588L, E638G,and A730V. In embodiments, the polymerase includes, relative to SEQ IDNO:1, F75L, R97C, I142T, V278I, A281T, A292E, M329L, P372S, H440N,E563G, E579G, F588L, D729Y, and V742A. In embodiments, the polymeraseincludes, relative to SEQ ID NO:1, I65V, R97H, F283S, V308M, K465E,F588L, T591A, I604M, G635D, and K727T. In embodiments, the polymeraseincludes, relative to SEQ ID NO:1, E25K, R97C, A168T, R255H, F326Y,K478R, R526H, E556K, K558N, P573S, E581N, F588L, E600K, E601K, R686H,R724H, V742A, K752E, and K762N. In embodiments, the polymerase includes,relative to SEQ ID NO:1, Y7H, K13I, F75L, R97L, I160V, V170I, A316P,K469E, A491V, E579G, F588L, V637D, H726D, and V742A. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, Y7H, K13R, A40V, R97H,F116L, A117S, K154E, A281T, I415V, K477I, K552N, N569S, E577K, A585G,F588I, E655D, and V742A. In embodiments, the polymerase includes,relative to SEQ ID NO:1, Y7H, K13R, I38F, A40V, R97C, D141A, P286L,G350S, R467C, and K469T, E579G, F588L, F721Y, H726D, V742A, L756C,R757A, W758G, Q759R, T761P, K762N, Q763R, V764L, and G765V. Inembodiments, the polymerase includes, relative to SEQ ID NO:1, V63A,F75L, R97C, D98G, R188H, F214I, G245S, D246E, T319I, G350S, E579G,F588L, R690H, H726D, V742A, and K760N. In embodiments, the polymeraseincludes, relative to SEQ ID NO:1, Y7H, I18V, R97C, A168S, F214S, G284S,P286Q, A292V, G304D, K391I, E431D, K477I, Y567H, E579G, F588L, andH726D. In embodiments, the polymerase includes, relative to SEQ ID NO:1,Y7H, IBV, N23D, V66A, F75L, R97C, P217Q, A298S, A316P, K465E, R526H,E577K, E601D, T606I, E655D, R706C, V742A, and W769S. In embodiments, thepolymerase includes, relative to SEQ ID NO:1, Y7H, K13R, L76P, K192E,K289T, R364C, L397M, Q484H, E563G, F588L, V637D, R706H, V742A, andL766P.

In embodiments, the polymerase includes an amino acid sequence that isat least 85% identical to a continuous 500 amino acid sequence withinSEQ ID NO: 1. In embodiments, the polymerase includes an amino acidsequence that is at least 90% identical to a continuous 500 amino acidsequence within SEQ ID NO: 1. In embodiments, the polymerase includes anamino acid sequence that is at least 95% identical to a continuous 500amino acid sequence within SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is at least 98% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1. In embodiments,the polymerase includes an amino acid sequence that is at least 99%identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.In embodiments, the polymerase includes an amino acid sequence that is90% identical to a continuous 500 amino acid sequence within SEQ IDNO: 1. In embodiments, the polymerase includes an amino acid sequencethat is 95% identical to a continuous 500 amino acid sequence within SEQID NO: 1. In embodiments, the polymerase includes an amino acid sequencethat is 90% identical to SEQ ID NO: 1. In embodiments, the polymeraseincludes an amino acid sequence that is 95% identical to SEQ ID NO: 1.

In embodiments, the polymerase (e.g., a polymerase as described herein)is truncated at the C-terminus. In embodiments, the polymerase (e.g., apolymerase as described herein) is truncated at the C-terminus andretains the ability to incorporate a modified nucleotide. Inembodiments, the polymerase (e.g., a polymerase as described herein) istruncated at the C-terminus, wherein the polymerase is truncated toremove at least 20 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the polymerase is truncated to remove at least 10amino acids from the C-terminus. In embodiments, the polymerase (e.g., apolymerase as described herein) is truncated at the C-terminus, whereinthe polymerase is truncated to remove at least 5 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 5 to 16 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the truncation removes 5 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 10 amino acids from the C-terminus. In embodiments, thepolymerase (e.g., a polymerase as described herein) is truncated at theC-terminus, wherein the truncation removes 13 amino acids from theC-terminus. In embodiments, the polymerase (e.g., a polymerase asdescribed herein) is truncated at the C-terminus, wherein the truncationremoves 16 amino acids from the C-terminus.

In embodiments, the polymerase includes a polycationic sequence (e.g., apolyhistidine tag, such as a His-6 tag). To facilitate synthesis and/orpurification, in embodiments a His6 tag (i.e., six consecutive histidineamino acids) are ligated to the C or N terminus of the polypeptidechain. It is understood that the presence of a His6 tag enables theisolation of peptide or protein products directly from ligation reactionmixtures by Ni-NTA affinity column purification. For example, commonpolyhistidine tags are formed of six histidine (6×His tag) residueswhich are added at the N-terminus preceded by methionine or C-terminusbefore a stop codon. Alternative polycationic sequences includealternating histidine and glutamine (e.g., three sets of HQ, referred toas an HQ tag) or alternating histidine and asparagine (e.g., six sets ofHN, referred to as an HN tag).

In another aspect is provided a nucleic acid encoding a mutant orimproved DNA polymerase as described herein, a vector comprising therecombinant nucleic acid, and/or a host cell transformed with thevector. In certain embodiments, the vector is an expression vector. Hostcells comprising such expression vectors are useful in methods of theinvention for producing the mutant or improved polymerase by culturingthe host cells under conditions suitable for expression of therecombinant nucleic acid. The polymerases of the invention may becontained in reaction mixtures and/or kits. The embodiments of therecombinant nucleic acids, host cells, vectors, expression vectors,reaction mixtures and kits are as described herein. The full plasmidnucleic acid sequence used to generate a polymerase is provided in SEQID NO: 2.

In an aspect is provided a kit, wherein the kit includes a polymerase asdescribed herein. Generally, the kit includes one or more containersproviding a composition, and one or more additional reagents (e.g., abuffer suitable for polynucleotide extension). The kit may also includea template nucleic acid (DNA and/or RNA), one or more primerpolynucleotides, nucleotides (including, e.g., deoxyribonucleotides,ribonucleotides, labeled nucleotides, and/or modified nucleotides),buffers, salts, and/or labels (e.g., fluorophores). In embodiments, thekit further includes instructions. In embodiments the kit includes oneor more enclosures (e.g., boxes, bottles, or cartridges) containing therelevant reaction reagents and/or supporting materials.

Adapters and/or primers may be supplied in the kits ready for use, asconcentrates-requiring dilution before use, or in a lyophilized or driedform requiring reconstitution prior to use. If required, the kits mayfurther include a supply of a suitable diluent for dilution orreconstitution of the primers and/or adapters. Optionally, the kits mayfurther include supplies of reagents, buffers, enzymes, and dNTPs foruse in carrying out nucleic acid amplification and/or sequencing.Further components which may optionally be supplied in the kit includesequencing primers suitable for sequencing templates prepared using themethods described herein.

In embodiments, kits described herein include a polymerase. Inembodiments, the polymerase is a DNA polymerase. In embodiments, the DNApolymerase is a thermophilic nucleic acid polymerase. In embodiments,the DNA polymerase is a modified archaeal DNA polymerase. Inembodiments, the kit includes a buffered solution. Typically, thebuffered solutions contemplated herein are made from a weak acid and itsconjugate base or a weak base and its conjugate acid. For example,sodium acetate and acetic acid are buffer agents that can be used toform an acetate buffer. Other examples of buffer agents that can be usedto make buffered solutions include, but are not limited to, Tris,bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, otherbuffer agents that can be used in enzyme reactions, hybridizationreactions, and detection reactions are known in the art. In embodiments,the buffered solution can include Tris. With respect to the embodimentsdescribed herein, the pH of the buffered solution can be modulated topermit any of the described reactions. In some embodiments, the bufferedsolution can have a pH greater than pH 7.0, greater than pH 7.5, greaterthan pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, orgreater than pH 11.5. In other embodiments, the buffered solution canhave a pH ranging, for example, from about pH 6 to about pH 9, fromabout pH 8 to about pH 10, or from about pH 7 to about pH 9. Inembodiments, the buffered solution can include one or more divalentcations. Examples of divalent cations can include, but are not limitedto, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. In embodiments, the buffered solutioncan contain one or more divalent cations at a concentration sufficientto permit hybridization of a nucleic acid.

III. Methods

In an aspect, a method of incorporating a nucleotide into a nucleic acidsequence is provided. The method includes allowing the followingcomponents to interact (e.g., by combining in a reaction vessel undersuitable conditions): (i) a nucleic acid template, (ii) a primer thathas an extendible 3′ end, (iii) a nucleotide solution, and (iv) apolymerase (e.g., a DNA polymerase or a thermophilic nucleic acidpolymerase as described herein). The polymerase used in the methodincludes an amino acid sequence that is at least 80% identical to acontinuous 500 amino acid sequence within SEQ ID NO: 1 and includes oneor more of the mutations described herein.

In an aspect is provided a method of incorporating a nucleotide into anucleic acid sequence including combining in a reaction vessel: (i) anucleic acid template, (ii) a nucleotide solution, and (iii) apolymerase, wherein the polymerase is a polymerase as described herein.In embodiments, the method includes combining the components in areaction vessel under conditions for incorporating and/orpolymerization. Such conditions are known in the art and describedherein.

In embodiments, the method includes amplifying the polynucleotide via anisothermal amplification process. Isothermal amplification processesinclude SDA, LAMP, SMAP, ICAN, SMART. In these techniques, the extensionreaction proceeds at a constant temperature, for example using stranddisplacement reactions. Amplification can be completed in a single step,by incubating the mixture of samples, primers, DNA polymerase withstrand displacement activity, and substrates at a constant temperature.This reduces the number of steps required, eliminating thermal rampingsteps and reducing the total cycle time for each amplification cycle,while simultaneously decreasing the reaction time required for eachcycle.

In another aspect is provided a method of amplifying a nucleic acidsequence including: a. hybridizing a nucleic acid template with a primerto form a primer-template hybridization complex; b. contacting theprimer-template hybridization complex with a DNA polymerase andnucleotides (e.g., a plurality of nucleotides, such as nativenucleotides), wherein the DNA polymerase is the polymerase as describedherein; and c. subjecting the primer-template hybridization complex toconditions which enable the polymerase to incorporate one or morenucleotides into the primer-template hybridization complex to generateamplification products, thereby amplifying a nucleic acid sequence.

In embodiments, the amplification products are double-strandedamplification products. In embodiments, generating a double-strandedamplification product includes amplifying the template polynucleotide orcomplement thereof on a solid support including a plurality of primersattached to the solid support, wherein the plurality of primers includea plurality of forward primers with complementarity to the templatepolynucleotide and a plurality of reverse primers with complementarityto a complement of the template polynucleotide, and the amplifyingincludes a plurality of cycles of strand denaturation, primerhybridization, and primer extension.

In embodiments, the plurality of strand denaturation cycles aredifferent for one or more cycles, wherein the initial denaturation cycleis maintained at different conditions from the remaining denaturationcycles. For example, in embodiments, the initial denaturation cycle isat about 85° C.-95° C. for about 1 minute to about 10 minutes, whereasdenaturation in the remaining cycles is different (e.g., about 85° C.for about 15-30 sec). In embodiments, the initial denaturation ismaintained at about 85° C.-95° C. for about 5 minutes to about 10minutes. In embodiments, the initial denaturation is maintained at 90°C.-95° C. for about 1 to 10 minutes. In embodiments, the initialdenaturation is maintained at 80° C.-85° C. for about 1 to 10 minutes.In embodiments, the initial denaturation is maintained at 85° C.-90° C.for about 1 to 10 minutes. In embodiments, the initial denaturation ismaintained at about 85° C.-95° C. for about 1 minutes to about 10minutes. In embodiments, the initial denaturation is maintained at about95° C. for about 5 minutes to about 10 minutes. In embodiments, theinitial denaturation is maintained at about 85° C.-95° C. for about 5minutes to about 10 minutes.

In embodiments, generating a double-stranded amplification productincludes a thermal bridge polymerase chain reaction (t-bPCR)amplification. In embodiments, the plurality of cycles includesthermally cycling between (i) about 85° C. for about 15-30 sec fordenaturation, and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 30 secondsfor annealing/extension of the primer.

In embodiments, the plurality of cycles includes thermally cyclingbetween (i) about 80° C. to 90° C. for denaturation, and (ii) about 55°C. to about 65° C. for annealing/extension of the primer. Inembodiments, the plurality of cycles includes thermally cycling between(i) about 85° C. for denaturation, and (ii) about 55° C. forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer. In embodiments, the plurality of cycles includes thermallycycling between (i) less than 80° C. (e.g., 70 to 80° C.) fordenaturation, and (ii) about 55° C. to about 65° C. forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 70° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer. In embodiments, the plurality of cycles includes thermallycycling between (i) about 75° C. for denaturation, and (ii) about 55° C.for annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. fordenaturation, and (ii) about 65° C. for annealing/extension of theprimer.

In embodiments, the plurality of cycles includes thermally cyclingbetween (i) about 85° C. for less than 1 minute for denaturation, and(ii) about 65° C. for about 1 to 2 minutes for annealing/extension ofthe primer. In embodiments, the plurality of cycles includes thermallycycling between (i) about 85° C. for less than 1 minute fordenaturation, and (ii) about 60° C. to about 65° C. for about 1 minutefor annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about 30sec for denaturation and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 30 secondsfor annealing/extension of the primer. In embodiments, the plurality ofcycles includes thermally cycling between (i) about 85° C. for about15-30 sec for denaturation, and (ii) about 65° C. for about 1 minute forannealing/extension of the primer. In embodiments, the temperature andduration for the annealing of the primer and the extension of the primerare different. In embodiments, the plurality of cycles includesthermally cycling between (i) about 90° C. to 95° C. for about 15 to 30sec for denaturation and (ii) about 55° C. to about 65° C. for about 30to 60 seconds for annealing and about 65° C. to 70° C. for about 30 to60 seconds for extension of the primer. In embodiments, the plurality ofdenaturation steps is at a temperature of about 80° C.-95° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 80° C.-90° C. In embodiments, the plurality of denaturation stepsis at a temperature of about 85° C.-90° C. In embodiments, the pluralityof denaturation steps is at a temperature of about 81° C., 82° C., 83°C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., or about 90° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., orabout 99° C. In embodiments, the plurality of denaturation steps is at atemperature of about 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93°C., 94° C., or about 95° C. In embodiments, the plurality ofdenaturation steps is at a temperature of about 90° C., 91° C., 92° C.,93° C., 94° C., or about 95° C. In embodiments, the plurality ofdenaturation steps is at a temperature of about 70° C.-85° C. Inembodiments, the plurality of denaturation steps is at a temperature ofabout 70° C.-80° C. In embodiments, the plurality of denaturation stepsis at a temperature of about 75° C.-80° C. In embodiments, the pluralityof denaturation steps is at a temperature of about 70° C., 71° C., 72°C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., or about 80°C. In embodiments, the annealing/extension of the primer cycle is at atemperature of about 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61°C., 62° C., 63° C., 64° C., or about 65° C.

In embodiments, the method includes includes exponential rolling circleamplification (eRCA). Exponential RCA is similar to the linear processexcept that it uses a second primer having a sequence that is identicalto at least a portion of the circular template (Lizardi et al. Nat.Genet. 19:225 (1998)). This two-primer system achieves isothermal,exponential amplification. Exponential RCA has been applied to theamplification of non-circular DNA through the use of a linear probe thatbinds at both of its ends to contiguous regions of a target DNA followedby circularization using DNA ligase (Nilsson et al. Science265(5181):208 5(1994)).

In embodiments, forming a plurality of amplification products includeshyperbranched rolling circle amplification (HRCA). Hyperbranched RCAuses a second primer complementary to the first amplification product.This allows products to be replicated by a strand-displacementmechanism, which can yield a drastic amplification within an isothermalreaction (Lage et al., Genome Research 13:294-307 (2003), which isincorporated herein by reference in its entirety).

In embodiments, the method includes amplifying a template nucleic acidby extending an amplification primer with a strand-displacing polymerasefor about 10 seconds to about 30 minutes. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase for about 30seconds to about 16 minutes. In embodiments, the method includesamplifying a template nucleic acid by extending an amplification primerwith a strand-displacing polymerase for about 30 seconds to about 10minutes. In embodiments, the method includes amplifying a templatenucleic acid by extending an amplification primer with astrand-displacing polymerase for about 30 seconds to about 5 minutes. Inembodiments, the method includes amplifying a template nucleic acid byextending an amplification primer with a strand-displacing polymerasefor about 1 second to about 5 minutes. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase for about 1second to about 2 minutes.

In embodiments, the method includes amplifying a template nucleic acidby extending an amplification primer with a strand-displacing polymeraseat a temperature of about 20° C. to about 50° C. In embodiments, themethod includes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase at atemperature of about 30° C. to about 50° C. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase at atemperature of about 25° C. to about 45° C. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase at atemperature of about 35° C. to about 45° C. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase at atemperature of about 35° C. to about 42° C. In embodiments, the methodincludes amplifying a template nucleic acid by extending anamplification primer with a strand-displacing polymerase at atemperature of about 37° C. to about 40° C.

In embodiments, the nucleic acid template includes one or more adapters.The term “adapter” as used herein refers to any oligonucleotide that canbe ligated to a nucleic acid molecule, thereby generating nucleic acidproducts that can be sequenced on a sequencing platform (e.g., anIllumina or Singular Genomics G4™ sequencing platform). In embodiments,adapters include two reverse complementary oligonucleotides forming adouble-stranded structure. In embodiments, an adapter includes twooligonucleotides that are complementary at one portion and mismatched atanother portion, forming a Y-shaped or fork-shaped adapter that isdouble stranded at the complementary portion and has two overhangs atthe mismatched portion. Since Y-shaped adapters have a complementary,double-stranded region, they can be considered a special form ofdouble-stranded adapters. When this disclosure contrasts Y-shapedadapters and double stranded adapters, the term “double-strandedadapter” or “blunt-ended” is used to refer to an adapter having twostrands that are fully complementary, substantially (e.g., more than 90%or 95%) complementary, or partially complementary. In embodiments,adapters include sequences that bind to sequencing primers. Inembodiments, adapters include sequences that bind to immobilizedoligonucleotides (e.g., P7 and P5 sequences) or reverse complementsthereof. In embodiments, the adapter is substantially non-complementaryto the 3′ end or the 5′ end of any target polynucleotide present in thesample. In embodiments, the adapter can include a sequence that issubstantially identical, or substantially complementary, to at least aportion of a primer, for example a universal primer. In embodiments, theadapter can include an index sequence (also referred to as barcode ortag) to assist with downstream error correction, identification orsequencing. In some embodiments, an adapter is hairpin adapter. In someembodiments, a hairpin adapter comprises a single nucleic acid strandcomprising a stem-loop structure. In some embodiments, a hairpin adaptercomprises a nucleic acid having a 5′-end, a 5′-portion, a loop, a3′-portion and a 3′-end (e.g., arranged in a 5′ to 3′ orientation). Insome embodiments, the 5′ portion of a hairpin adapter is annealed and/orhybridized to the 3′ portion of the hairpin adapter, thereby forming astem portion of the hairpin adapter. In some embodiments, the 5′ portionof a hairpin adapter is substantially complementary to the 3′ portion ofthe hairpin adapter. In certain embodiments, a hairpin adapter comprisesa stem portion (i.e., stem) and a loop, wherein the stem portion issubstantially double stranded thereby forming a duplex. In someembodiments, the loop of a hairpin adapter comprises a nucleic acidstrand that is not complementary (e.g., not substantially complementary)to itself or to any other portion of the hairpin adapter. In someembodiments, a method herein comprises ligating a first adapter to afirst end of a double stranded nucleic acid, and ligating a secondadapter to a second end of a double stranded nucleic acid. In someembodiments, the first adapter and the second adapter are different. Forexample, in certain embodiments, the first adapter and the secondadapter may comprise different nucleic acid sequences or differentstructures. In some embodiments, the first adapter is a Y-adapter andthe second adapter is a hairpin adapter. In some embodiments, the firstadapter is a hairpin adapter and a second adapter is a hairpin adapter.In certain embodiments, the first adapter and the second adapter maycomprise different primer binding sites, different structures, and/ordifferent capture sequences (e.g., a sequence complementary to a capturenucleic acid). In some embodiments, some, all or substantially all ofthe nucleic acid sequence of a first adapter and a second adapter arethe same. In some embodiments, some, all or substantially all of thenucleic acid sequence of a first adapter and a second adapter aresubstantially different.

In embodiments, the nucleic acid template includes common sequences attheir 5′ and 3′ ends. In this context the term “common” is interpretedas meaning common to all templates in the library. For example, thedouble-stranded amplification product may include a first adaptersequence at the 5′ end and a second adapter sequence at the 3′ end.Typically, the first adapter sequence and the second adapter sequencewill consist of no more than 100, or no more than 50, or no more than 40consecutive nucleotides at the 5′ and 3′ ends, respectively, of eachstrand of each template polynucleotide. The precise length of the twosequences may or may not be identical. The precise sequences of thecommon regions are generally not material to the invention and may beselected by the user. The common sequences must at least includeprimer-binding sequences (i.e., regions of complementarity for a primer)which enable specific annealing of primers when the templatepolynucleotides are in used in a solid-phase amplification reaction. Theprimer-binding sequences are thus determined by the sequence of theprimers to be ultimately used for solid-phase amplification.

In embodiments, the method includes amplifying the templatepolynucleotide in a cell. In embodiments, the method includes amplifyingthe template polynucleotide in a tissue. In embodiments, the methodincludes amplifying the template polynucleotide one a solid support(e.g., a multiwell container). In embodiments, the amplification primeris immobilized on a solid support.

A nucleic acid can be amplified by a suitable method. The term“amplified” as used herein refers to subjecting a target nucleic acid ina sample to a process that linearly or exponentially generates ampliconnucleic acids having the same or substantially the same (e.g.,substantially identical) nucleotide sequence as the target nucleic acid,or segment thereof, and/or a complement thereof. In some embodiments anamplification reaction comprises a suitable thermal stable polymerase.Thermal stable polymerases (e.g., a polymerase described herein) arestable for prolonged periods of time, at temperature greater than 80° C.when compared to common polymerases found in most mammals. In certainembodiments the term “amplified” refers to a method that comprises apolymerase chain reaction (PCR). Conditions conducive to amplification(i.e., amplification conditions) are known and often comprise at least asuitable polymerase (e.g., a polymerase as described herein), a suitabletemplate, a suitable primer or set of primers, suitable nucleotides(e.g., dNTPs), a suitable buffer, and application of suitable annealing,hybridization and/or extension times and temperatures. In certainembodiments an amplified product (e.g., an amplicon) can contain one ormore additional and/or different nucleotides than the template sequence,or portion thereof, from which the amplicon was generated (e.g., aprimer can contain “extra” nucleotides (such as a 5′ portion that doesnot hybridize to the template), or one or more mismatched bases within ahybridizing portion of the primer).

A nucleic acid can be amplified by a thermocycling method or by anisothermal amplification method. In some embodiments, a rolling circleamplification method is used. In some embodiments, amplification takesplace on a solid support (e.g., within a flow cell) where a nucleicacid, nucleic acid library or portion thereof is immobilized. In certainsequencing methods, a nucleic acid library is added to a flow cell andimmobilized by hybridization to anchors under suitable conditions. Thistype of nucleic acid amplification is often referred to as solid phaseamplification. In some embodiments of solid phase amplification, all ora portion of the amplified products are synthesized by an extensioninitiating from an immobilized primer. Solid phase amplificationreactions are analogous to standard solution phase amplifications exceptthat at least one of the amplification oligonucleotides (e.g., primers)is immobilized on a solid support.

In some embodiments solid phase amplification comprises a nucleic acidamplification reaction comprising only one species of oligonucleotideprimer immobilized to a surface or substrate. In certain embodimentssolid phase amplification comprises a plurality of different immobilizedoligonucleotide primer species. In some embodiments solid phaseamplification may comprise a nucleic acid amplification reactioncomprising one species of oligonucleotide primer immobilized on a solidsurface and a second different oligonucleotide primer species insolution. Multiple different species of immobilized or solution-basedprimers can be used. Non-limiting examples of solid phase nucleic acidamplification reactions include interfacial amplification, bridgeamplification, emulsion PCR, WildFire amplification (e.g., US patentpublication US20130012399), the like or combinations thereof.

In embodiments, the nucleic acid template is DNA, RNA, or analogsthereof. In embodiments, the nucleic acid template includes a primerhybridized to the template. In embodiments, the nucleic acid template isa primer. Primers are usually single-stranded for maximum efficiency inamplification, but may alternatively be double-stranded. Ifdouble-stranded, the primer is usually first treated to separate itsstrands before being used to prepare extension products. Thisdenaturation step is typically affected by heat, but may alternativelybe carried out using alkali, followed by neutralization. Thus, a“primer” is complementary to a nucleic acid template, and complexes byhydrogen bonding or hybridization with the template to give aprimer/template complex for initiation of synthesis by a polymerase,which is extended by the addition of covalently bonded bases linked attheir 3′ end complementary to the template in the process of DNAsynthesis. The DNA template for a sequencing reaction will typicallycomprise a double-stranded region having a free 3′ hydroxyl group whichserves as a primer or initiation point for the addition of furthernucleotides in the sequencing reaction. The region of the DNA templateto be sequenced will overhang this free 3′ hydroxyl group on thecomplementary strand. The primer bearing the free 3′ hydroxyl group maybe added as a separate component (e.g. a short oligonucleotide), whichhybridizes to a region of the template to be sequenced. Alternatively,the primer and the template strand to be sequenced may each form part ofa partially self-complementary nucleic acid strand capable of forming anintramolecular duplex, such as for example a hairpin loop structure.Nucleotides are added successively to the free 3′ hydroxyl group,resulting in synthesis of a polynucleotide chain in the 5′ to 3′direction. After each nucleotide addition the nature of the base whichhas been added will be determined, thus providing sequence informationfor the DNA template.

In embodiments, the template polynucleotide includes genomic DNA,complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA),transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), ornoncoding RNA (ncRNA).

In embodiments, the template polynucleotide is about 100 to 1000nucleotides in length. In embodiments, the template polynucleotide isabout 500 to 2000 nucleotides in length. In embodiments, the templatepolynucleotide is about 1000 to 1000 nucleotides in length. Inembodiments, the template polynucleotide is about 50 to 500 nucleotidesin length. In embodiments, the template polynucleotide is about 500 to1000 nucleotides in length. In embodiments, the template polynucleotideis about 350 nucleotides in length. In embodiments, the templatepolynucleotide is about 10, 20, 50, 100, 150, 200, 300, or 500nucleotides in length. The template polynucleotide molecules can varylength, such as about 100-300 nucleotides long, about 300-500nucleotides long, or about 500-1000 nucleotides long. In embodiments,the template polynucleotide molecular is about 100-1000 nucleotides,about 150-950 nucleotides, about 200-900 nucleotides, about 250-850nucleotides, about 300-800 nucleotides, about 350-750 nucleotides, about400-700 nucleotides, or about 450-650 nucleotides. In embodiments, thetemplate polynucleotide molecule is about 150 nucleotides. Inembodiments, the template polynucleotide is about 100-1000 nucleotideslong. In embodiments, the template polynucleotide is about 100-300nucleotides long. In embodiments, the template polynucleotide is about300-500 nucleotides long. In embodiments, the template polynucleotide isabout 500-1000 nucleotides long. In embodiments, the templatepolynucleotide molecule is about 100 nucleotides. In embodiments, thetemplate polynucleotide molecule is about 300 nucleotides. Inembodiments, the template polynucleotide molecule is about 500nucleotides. In embodiments, the template polynucleotide molecule isabout 1000 nucleotides.

In embodiments the template polynucleotide (e.g., genomic template DNA)is first treated to form single-stranded linear fragments (e.g., rangingin length from about 50 to about 600 nucleotides). Treatment typicallyentails fragmentation, such as by chemical fragmentation, enzymaticfragmentation, or mechanical fragmentation, followed by denaturation toproduce single-stranded DNA fragments. In embodiments, the templatepolynucleotide includes an adapter. The adapter may have otherfunctional elements including tagging sequences (i.e., a barcode),attachment sequences, palindromic sequences, restriction sites,sequencing primer binding sites, functionalization sequences, and thelike. Barcodes can be of any of a variety of lengths. In embodiments,the primer includes a barcode that is 10-50, 20-30, or 4-12 nucleotidesin length. In embodiments, the adapter includes a primer bindingsequence that is complementary to at least a portion of a primer (e.g.,a sequencing primer). Primer binding sites can be of any suitablelength. In embodiments, a primer binding site is about or at least about10, 15, 20, 25, 30, or more nucleotides in length. In embodiments, aprimer binding site is 10-50, 15-30, or 20-25 nucleotides in length.

In embodiments, the template polynucleotide and the double-strandedamplification products include known adapter sequences on the 5′ and 3′ends. In embodiments, the template polynucleotide includes known adaptersequences on the 5′ and 3′ ends. In embodiments, the double-strandedamplification products include known adapter sequences on the 5′ and 3′ends.

In some embodiments, the reaction conditions for amplification includesincubation in a denaturant. As used herein, the terms “denaturant” orplural “denaturants” are used in accordance with their plain andordinary meanings and refer to an additive or condition that disruptsthe base pairing between nucleotides within opposing strands of adouble-stranded polynucleotide molecule. The term “denature” and itsvariants, when used in reference to any double-stranded polynucleotidemolecule, or double-stranded polynucleotide sequence, includes anyprocess whereby the base pairing between nucleotides within opposingstrands of the double-stranded molecule, or double-stranded sequence, isdisrupted. Typically, denaturation includes rendering at least someportion or region of two strands of the double-stranded polynucleotidemolecule or sequence single-stranded or partially single-stranded. Insome embodiments, denaturation includes separation of at least someportion or region of two strands of the double-stranded polynucleotidemolecule or sequence from each other.

Typically, the denatured region or portion is then capable ofhybridizing to another polynucleotide molecule or sequence. Optionally,there can be “complete” or “total” denaturation of a double-strandedpolynucleotide molecule or sequence. Complete denaturation conditionsare, for example, conditions that would result in complete separation ofa significant fraction (e.g., more than 10%, 20%, 30%, 40% or 50%) of alarge plurality of strands from their extended and/or full-lengthcomplements. Typically, complete or total denaturation disrupts all ofthe base pairing between the nucleotides of the two strands with eachother. Similarly, a nucleic acid sample is optionally considered fullydenatured when more than 80% or 90% of individual molecules of thesample lack any double-strandedness (or lack any hybridization to acomplementary strand).

Alternatively, the double-stranded polynucleotide molecule or sequencecan be partially or incompletely denatured. A given nucleic acidmolecule can be considered partially denatured when a portion of atleast one strand of the nucleic acid remains hybridized to acomplementary strand, while another portion is in an unhybridized state(even if it is in the presence of a complementary sequence). Theunhybridized portion is optionally at least 5, 10, 15, 20, 50, or morenucleotides in length. The hybridized portion is optionally at least 5,10, 15, 20, 50, or more nucleotides in length. Partial denaturationincludes situations where some, but not all, of the nucleotides of onestrand or sequence, are based paired with some nucleotides of the otherstrand or sequence within a double-stranded polynucleotide. In someembodiments, at least 20% but less than 100% of the nucleotide residuesof one strand of the partially denatured polynucleotide (or sequence)are not base paired to nucleotide residues within the opposing strand.In embodiments, at least 50% of nucleotide residues within thedouble-stranded polynucleotide molecule (or double-strandedpolynucleotide sequence) are in single-stranded (or unhybridized) from,but less than 20% or 10% of the residues are double-stranded.

Optionally, a nucleic acid sample can be considered to be partiallydenatured when a substantial fraction of individual nucleic acidmolecules of the sample (e.g., above 20%, 30%, 50%, or 70%) are in apartially denatured state. Optionally less than a substantial amount ofindividual nucleic acid molecules in the sample are fully denatured,e.g., not more than 5%, 10%, 20%, 30% or 50% of the nucleic acidmolecules in the sample. Under exemplary conditions at least 50% of thenucleic acid molecules of the sample are partly denatured, but less than20% or 10% are fully denatured. In other situations, at least 30% of thenucleic acid molecules of the sample are partly denatured, but less than10% or 5% are fully denatured. Similarly, a nucleic acid sample can benon-denatured when a minority of individual nucleic acid molecules inthe sample are partially or completely denatured.

In an embodiment, partially denaturing conditions are achieved bymaintaining the duplexes as a suitable temperature range. For example,the nucleic acid is maintained at temperature sufficiently elevated toachieve some heat-denaturation (e.g., above 45° C., 50° C., 55° C., 60°C., 65° C., or 70° C.) but not high enough to achieve completeheat-denaturation (e.g., below 95° C. or 90° C. or 85° C. or 80° C. or75° C.). In an embodiment the nucleic acid is partially denatured usingsubstantially isothermal conditions. Alternatively, chemicaldenaturation can be accomplished by contacting the double-strandedpolynucleotide to be denatured with appropriate chemical denaturants,such as strong alkalis, strong acids, chaotropic agents, and the likeand can include, for example, NaOH, urea, or guanidine-containingcompounds. In some embodiments, partial or complete denaturation isachieved by exposure to chemical denaturants such as urea or formamide,with concentrations suitably adjusted, or using high or low pH (e.g., pHbetween 4-6 or 8-9). In embodiments, the denaturant is a bufferedsolution including betaine, dimethyl sulfoxide (DMSO), ethylene glycol,formamide, glycerol, guanidine thiocyanate, 4-methylmorpholine 4-oxide(NMO), or a mixture thereof. In embodiments, the first denaturant is abuffered solution including about 0% to about 50% dimethyl sulfoxide(DMSO); about 0% to about 50% ethylene glycol; about 0% to about 20%formamide; or about 0 to about 3M betaine, or a mixture thereof. In anembodiment herein, partial denaturation and/or amplification, includingany one or more steps or methods described herein, can be achieved usinga recombinase and/or single-stranded binding protein.

In some embodiments, complete or partial denaturation of adouble-stranded polynucleotide sequence is accomplished by contactingthe double-stranded polynucleotide sequence using appropriate denaturingagents. For example, the double-stranded polynucleotide can be subjectedto heat-denaturation (also referred to interchangeably as thermaldenaturation) by raising the temperature to a point where the desiredlevel of denaturation is accomplished. In some embodiments, thermaldenaturation of a double-stranded polynucleotide, includes adjusting thetemperature to achieve complete separation of the two strands of thepolynucleotide, such that 90% or greater of the strands are insingle-stranded form across their entire length. A completely denatureddouble-stranded polynucleotide results in a separated first strand and asecond strand, each of which is a single-stranded polynucleotide. Insome embodiments, complete thermal denaturation of a polynucleotidemolecule (or polynucleotide sequence) is accomplished by exposing thepolynucleotide molecule (or sequence) to a temperature that is at least5° C., 10° C., 15° C., 20° C., 25° C., 30° C., 50° C., or 100° C., abovethe calculated or predict melting temperature (Tm) of the polynucleotidemolecule or sequence.

In some embodiments, complete or partial denaturation is accomplished bytreating the double-stranded polynucleotide sequence to be denaturedusing a denaturant mixture including an SSB protein (e.g., T4 gp32protein, T7 gene 2.5 SSB protein, or phi29 SSB protein, Thermococcuskodakarensis (KOD) SSB, Therms thermophilus (TTH) SSB, Sulfolobussolfataricus (SSO) SSB, or Extreme Thermostable Single-Stranded DNABinding Protein (ET-SSB)), a strand-displacing polymerase (e.g., Bstlarge fragment (Bst LF) polymerase, Bst 3.0 polymerase, Bst 2.0polymerase, Bsu polymerase, SD polymerase, Vent exo-polymerase, Phi29polymerase, or a mutant thereof), and one or more crowding agents(poly(ethylene glycol) (PEG), polyvinylpyrrolidone (PVP), bovine serumalbumin (BSA), dextran, Ficoll (e.g., Ficoll 70 or Ficoll 400),glycerol, or a combination thereof). In embodiments, the crowding agentis poly(ethylene glycol) (e.g., PEG 200, PEG 600, PEG 800, PEG 2,050,PEG 4,600, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, or PEG 35,000),dextran sulfate, bovine pancreatic trypsin inhibitor (BPTI),ribonuclease A, lysozyme, β-lactoglobulin, hemoglobin, bovine serumalbumin (BSA), or poly(sodium 4-styrene sulfonate) (PSS). Inembodiments, the denaturant mixture including an SSB, astrand-displacing polymerase, and one or more crowding agents does notinclude a chemical denaturant (e.g., betaine, DMSO, ethylene glycol,formamide, guanidine thiocyanate, NMO, TMAC, or a mixture thereof).

In embodiments, mutation(s) may include substitution of the amino acidin the parent amino acid sequences with an amino acid, which is not theparent amino acid. In embodiments, the mutations may result inconservative amino acid changes. In embodiments, non-polar amino acidsmay be converted into polar amino acids (threonine, asparagine,glutamine, cysteine, tyrosine, aspartic acid, glutamic acid orhistidine) or the parent amino acid may be changed to an alanine.

In embodiments, the method includes maintaining the temperature at about55° C. In embodiments, the method includes maintaining the temperatureat about 55° C. to about 80° C. In embodiments, the method includesmaintaining the temperature at about 60° C. to about 70° C. Inembodiments, the method includes maintaining the temperature at about65° C. to about 75° C. In embodiments, the method includes maintainingthe temperature at about 65° C. In embodiments, the method includesmaintaining the temperature at about 60° C. In embodiments, the methodincludes maintaining the temperature at a pH of 8.0 to 11.0. Inembodiments, the pH is 9.0 to 11.0. In embodiments, the pH is 9.5. Inembodiments, the pH is 10.0. In embodiments, the pH is 8.0, 8.5, 9.0,9.5, 10.0, 10.5, or 11.0. In embodiments, the pH is from 9.0 to 11.0,and the temperature is about 60° C. to about 70° C. In embodiments, thepH is from 8.5 to 9.5, and the temperature is about 58° C. to about 62°C.

In embodiments of the methods and compositions provided herein, theclusters have a mean or median separation from one another of about0.5-5 μm. In embodiments, the mean or median separation is about 0.1-10microns, 0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a rangebetween any two of these values. In embodiments, the mean or medianseparation is about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4., 4.5, 4.6, 4.7, 4.8, 4.9,5.0 μm or a number or a range between any two of these values. Inembodiments, the mean or median separation is about 0.1-10 microns. Inembodiments, the mean or median separation is about 0.25-5 microns. Inembodiments, the mean or median separation is about 0.5-2 microns. Inembodiments, the mean or median separation is about or at least about0.1 μm. In embodiments, the mean or median separation is about or atleast about 0.25 μm. In embodiments, the mean or median separation isabout or at least about 0.5 μm. In embodiments, the mean or medianseparation is about or at least about 1.0 μm. In embodiments, the meanor median separation is about or at least about 2.0 μm. In embodiments,the mean or median separation is about or at least about 5.0 μm. Inembodiments, the mean or median separation is about or at least about 10μm. The mean or median separation may be measured center-to-center(i.e., the center of one cluster to the center of a second cluster). Inembodiments of the methods provided herein, the amplicon clusters have amean or median separation (measured center-to-center) from one anotherof about 0.5-5 μm. The mean or median separation may be measurededge-to-edge (i.e., the edge of one amplicon cluster to the edge of asecond amplicon cluster). In embodiments of the methods provided herein,the amplicon clusters have a mean or median separation (measurededge-to-edge) from one another of about 0.2-5 μm.

In embodiments of the methods provided herein, the amplicon clustershave a mean or median diameter of about 100-2000 nm, or about 200-1000nm. In embodiments, the mean or median diameter is about 100-3000nanometers, about 500-2500 nanometers, about 1000-2000 nanometers, or anumber or a range between any two of these values. In embodiments, themean or median diameter is about or at most about 100, 200, 300, 400,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2000 nanometers or a number or a rangebetween any two of these values. In embodiments, the mean or mediandiameter is about 100-3,000 nanometers. In embodiments, the mean ormedian diameter is about 100-2,000 nanometers. In embodiments, the meanor median diameter is about 500-2500 nanometers. In embodiments, themean or median diameter is about 200-1000 nanometers. In embodiments,the mean or median diameter is about 1,000-2,000 nanometers. Inembodiments, the mean or median diameter is about or at most about 100nanometers. In embodiments, the mean or median diameter is about or atmost about 200 nanometers. In embodiments, the mean or median diameteris about or at most about 500 nanometers. In embodiments, the mean ormedian diameter is about or at most about 400 nanometers. Inembodiments, the mean or median diameter is about or at most about 500nanometers. In embodiments, the mean or median diameter is about or atmost about 600 nanometers. In embodiments, the mean or median diameteris about or at most about 700 nanometers. In embodiments, the mean ormedian diameter is about or at most about 1,000 nanometers. Inembodiments, the mean or median diameter is about or at most about 2,000nanometers. In embodiments, the mean or median diameter is about or atmost about 2,500 nanometers. In embodiments, the mean or median diameteris about or at most about 3,000 nanometers.

In embodiments of the methods provided herein, each amplicon cluster(e.g., an amplicon cluster having a mean or median diameter of about100-2000 nm, or about 200-1000 nm) includes about or at least about 100,500, 1,000, 2,500, 5,000, 10,000, 15,000, 20,000, 25,000, 30,000,35,000, 40,000, 45,000, or 50,000 dsDNA molecules. In embodiments, eachamplicon cluster includes about 100 dsDNA molecules. In embodiments,each amplicon cluster includes about 500 dsDNA molecules. Inembodiments, each amplicon cluster includes about 1000 dsDNA molecules.In embodiments, each amplicon cluster includes about 500 dsDNAmolecules. In embodiments, each amplicon cluster includes about 1,000dsDNA molecules. In embodiments, each amplicon cluster includes about2,500 dsDNA molecules. In embodiments, each amplicon cluster includesabout 5,000 dsDNA molecules. In embodiments, each amplicon clusterincludes about 10,000 dsDNA molecules. In embodiments, each ampliconcluster includes about 20,000 dsDNA molecules. In embodiments, eachamplicon cluster includes about 30,000 dsDNA molecules. In embodiments,each amplicon cluster includes about 40,000 dsDNA molecules. Inembodiments, each amplicon cluster includes about 50,000 dsDNAmolecules. In embodiments, each amplicon cluster includes more thanabout 50,000 dsDNA molecules.

In embodiments, the substrate is a particle. In embodiments, thesubstrate is a multiwell container. In embodiments, the substrate is apolymer coated particle or polymer coated planar support. Inembodiments, the substrate includes a polymer. In embodiments, theparticle includes polymerized units of polyacrylamide (AAm),poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, sulfobetaineacrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholineacrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetainemethacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethyleneglycol acrylate, methacrylate, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA),hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA),hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof. In embodiments, the particleshell includes polymerized units of polyacrylamide (AAm), glicydylmethacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate(IEM), or a copolymer thereof. In embodiments, the particle includespolymerized units of polyethylene glycol methacrylate (PEGMA) andglicydyl methacrylate (GMA). In embodiments, the particle includespolymerized units of polyethylene glycol methacrylate (PEGMA) andisocyanatoethyl methacrylate (IEM). In embodiments, the particleincludes polymerized units of 3-azido-2-hydroxypropyl methacrylate,2-azido-3-hydroxypropyl methacrylate,2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle includes polymerized units of 3-azido-2-hydroxypropylmethacrylate, 2-azido-3-hydroxypropyl methacrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments,the particle includes polymerized units of 3-azido-2-hydroxypropylmethacrylate. In embodiments, the particle includes polymerized units of3-azido-2-hydroxypropyl methacrylate 2-azido-3-hydroxypropylmethacrylate. In embodiments, the particle includes polymerized units of3-azido-2-hydroxypropyl methacrylate2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.

In embodiments, the nucleic acid template or the nucleic acid primer areattached to a solid support. In embodiments, the solid support includesabout 100, 500, 1000, 5000, 10000, or more primer-template hybridizationcomplex (i.e., dsDNA molecules) in a 2 μm² area. In embodiments, thesolid support includes about 1,000 to about 10,000 dsDNA molecules in a2 μm² area. In embodiments, the solid support includes about 1,000 toabout 10,000 dsDNA molecules in a 0.5 μm diameter feature. Inembodiments, the solid support includes about 1,000 to about 50,000dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nm diameterfeature. In embodiments, the solid support includes about 10,000 toabout 50,000 dsDNA molecules in a 500, 600, 700, 800, 900, or 1,000 nmdiameter feature. In embodiments, the solid support includes about20,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800, 900, or1,000 nm diameter feature. In embodiments, the solid support includesabout 30,000 to about 40,000 dsDNA molecules in a 500, 600, 700, 800,900, or 1,000 nm diameter feature. As used herein, a feature may be awells, pits, channels, ridges, raised regions, pegs, or posts on a solidsupport. Each feature includes a colony and refers to a discrete site ona solid support that includes a plurality of immobilizedpolynucleotides.

In embodiments, the polymerases described herein have improvedpolymerase activity (i.e., improved relative to a control). Polymeraseactivity, in some instances, includes the measurable quantity k_(cat),k_(cat)/K_(m), or yields of incorporated nucleotides for a given timeperiod. In embodiments, the polymerases described herein have increasedextension activity (i.e., increased relative to a control). Increasedextension activity variously refers to an increase in reaction kinetics(increased k_(cat)), increased K_(D), decreased K_(m), increasedk_(cat)/K_(m) ratio, faster turnover rate, higher turnover number, orother metric that is beneficial to the use of the polypeptide fornucleic acid extension with nucleotides. The polypeptides describedherein often incorporate at least 30% more nucleotides than thewild-type polymerase in total or in a given duration of time.

In embodiments, the polymerases described herein often incorporate atleast 10%, 20%, 30%, 50%, 75%, 100%, 125%, 150%, 200%, 500%, morenucleotides than a control (e.g., the wild-type polymerase) for a fixedamount of time and same nucleotide concentration. In embodiments, thepolymerases described herein incorporate nucleotides at least 1.5, 2,2.5, 5, 10, 15, 20, 25, or at least 50 times faster than a control(e.g., the wild-type polymerase) for a fixed amount of time. Suchmeasurements are often measured under conditions such as a set period oftime, such as at least, at most, or exactly 1, 2, 3, 5, 8, 10, 15, 20,or more than 20 minutes. Such measurements are often measured underconditions such as a set nucleotide concentration, such as less than 10uM, 10 uM, 20 uM, 50 uM, 100 uM, 200 uM, 300 uM, 500 uM, or more than500 uM, or any concentration within the range identified herein

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1. Focused Mutational Analysis

DNA amplification has many applications in molecular biology researchand medical diagnostics. There are two main strategies for amplifying adefined sequence of nucleic acid: polymerase chain reaction (PCR) andisothermal amplification. The polymerase chain reaction relies uponthermal cycling to denature dsDNA templates, followed by annealingprimers at specific sites in the denatured template, and extension ofthe primers by a thermostable DNA polymerase. Isothermal amplificationof DNA, as the name implies, typically includes amplification of thedsDNA at a defined temperature. The lack of thermal cycling inisothermal amplification technologies reduces equipment needs andimproves the time to answer, especially for point-of-care applications.

A variety of isothermal amplification methods have been developed, forexample, strand displacement amplification (SDA) (Walker, G. T. et al.Nucleic Acids Res 20, 1691-6 (1992); and Walker, G. T., Little, M. C,Nadeau, J. G. & Shank, D. D. PNAS 89, 392-6 (1992)), rolling circleamplification (RCA) (Fire, A. & Xu, S. Q. PNAS 92, 4641-5 (1995)), crosspriming amplification (CPA) (Xu, G. et al. Sci. Rep. 2, 246; (2012)) andloop mediated amplification (LAMP) (Notomi, T. et al. Nucleic Acids Res28, e63 (2000)). While some isothermal amplification mechanisms dependupon multiple enzymes, e.g., nickases, recombinases, and ligases, toachieve continuous replication, RCA and LAMP require only polymerasesand primers. These methods, like many other isothermal amplificationmethods, require the use of a DNA polymerase with a strong stranddisplacement activity to displace downstream DNA, thereby enablingcontinuous replication without thermal cycling. Additionally, efficientamplification requires elevated temperatures (e.g., 60° C. or greater)to enable the annealing of primers at specific locations on the dsDNA.Thus, a DNA polymerase suitable for these methods must be a thermostableDNA polymerase with a strong strand displacement activity.

Few thermostable, strand displacing enzymes exist. For example, SD DNApolymerase (a mutant Taq DNA polymerase) and the large fragment of BstDNA polymerase possess favorable characteristics for isothermalamplification. Bacillus stearothermophilus (Bst) DNA Polymerase I is amember of polymerase family A and is one of the most popular enzymeswith strand displacement activity because its optimum is about 63° C.Unfortunately, Bst polymerase is limited to temperatures up to 68-70°C.; at temperature 68° C. or higher it is inactivated (Xu, G. et al.Sci. Rep. 2, 246; (2012)). This complicates the workflow, since manyisothermal amplification approaches depend on an initial heating step(e.g., to about 95° C.) to denature dsDNA templates. An aim of thegeneral experimental plan was to produce a robust, optimized polymerasefor nucleic acid sequencing methods. DNA polymerases of the Pyrococcusgenus share similar anerobic features as other thermophilic genera(e.g., Archaeoglobus, Thermoautotrophican, Methanococcus), however,Pyrococcus species thrive in higher temperatures, ca 100° C., andtolerate extreme pressures. For example, the area around undersea hotvents, where P. abyssi has been found, there is no sunlight, thetemperature is around 98° C.-100° C. and the pressure is about 200 atm.These Pyrococcus polymerases possess inherent properties that arebeneficial for sequencing applications.

Directed evolution of enzymes is a process that mimics natural selectionin vitro. Compartmentalized self-replication (CSR) is a method ofdirected evolution where a library containing mutated variants of theenzyme of interest goes through rounds of selective pressure, and overtime, the most active or best performing variants are enriched in thelibrary, compared to less active variants, as described in Abil, Z., &Ellington, A. D. (2018). Current Protocols in Chemical Biology, 10,1-17. During CSR, the enzyme variants and its own encoding genes arecompartmentalized in oil emulsions, together with dNTPs and primers.During the emulsion PCR, each enzyme that can surpass the selectivepressure is able to replicate its own encoding gene and pass to the nextround of selection. Over time, the best performers are enriched in thelibrary.

DNA polymerases carry out crucial functions in many DNA metabolicprocesses, and due to their ability to catalyze the replication of DNAby incorporating nucleotides into the 3′ end of a primer annealed to atemplate, DNA polymerases are frequently used in genomic research (e.g.,next-generation sequencing, or NGS, technologies). The human genomeencodes at least 14 DNA-dependent DNA polymerases, each serving aparticular function. The general classification includes five differentclasses according to their function: DNA polymerase (Pol α) catalyzesDNA replication at Okazaki fragments on the lagging strand; Pol βparticipates in base-excision repair; Pol γ is involved in mitochondrialDNA synthetic processes; Pol δ participates in lagging-strand synthesis;and Pol ε catalyzes the synthesis of the leading strand of chromosomalDNA.

Structural analyses of DNA polymerases portray the enzyme as analogousto a human right hand, with three domains: a ‘fingers’ domain thatinteracts with the incoming dNTP and paired template base, and thatcloses at each nucleotide addition step; a ‘palm’ domain that catalyzesthe phosphoryl-transfer reaction; and a ‘thumb’ domain that interactswith duplex DNA. The finger and palm subdomains of DNA polymerases(e.g., amino acids positions 448-603 of SEQ ID NO:1) are in closeproximity to the nucleotide incorporation region. We initially limitedour mutational analysis to mutations within the finger and palmsubdomains (i.e., examining mutations in amino acids positions 448-603of SEQ ID NO:1) before broadening out mutational analysis to the entireenzyme sequence (see, Example 2).

For brevity, amino acid mutation nomenclature is used throughout thisapplication. One having skill in the art would understand the amino acidmutation nomenclature, such that D141A refers to aspartic acid (singleletter code is D), at position 141, is replaced with alanine (singleletter code A). Likewise, it is understood that when an amino acidmutation nomenclature is used and the terminal amino acid code ismissing, e.g., P411, it is understood that no mutation was made relativeto the wild type. Additionally, for amino acid positions that arefrequently mutated herein, the wild type amino acid may be recited toemphasize that it is not mutated, for example P411P.

Prior to performing CSR, an error prone PCR library was generated with atarget error rate of 6 to 8 mutations per clone using a mutantsequencing enzyme having homology to the sequence SEQ ID NO:3. Themutations were restricted to the approximately 155 amino acids withinthe finger and palm subdomain, which corresponds to about 465nucleotides in the gene vector. The library was cloned using acommercial vector pET21b+ via Gibson Assembly, using the GibsonAssembly® Master Mix (NEB Catalog Number E2611S), using the standardprotocol described in Gibson, D. G. et. al. (2009) Nature Methods,343-345 and Gibson, D. G. et al. (2010) Nature Methods, 901-903. GibsonAssembly was developed by Dr. Daniel Gibson and colleagues at the J.Craig Venter Institute and licensed to NEB by Synthetic Genomics, Inc.It allows for successful assembly of multiple DNA fragments, regardlessof fragment length or end compatibility. Gibson Assembly efficientlyjoins multiple overlapping DNA fragments in a single-tube isothermalreaction. The end result is a double-stranded fully sealed DNA moleculethat can serve as template for PCR, RCA or a variety of other molecularbiology applications. The assembled plasmids containing the diverseinserts were then purified utilizing the Monarch® PCR & DNA Cleanup Kit(NEB Catalog Number T1030L), and then transformed into T7 ExpressElectrocompetent E. coli cells (NEB Catalog Number C3026J). Afterplating the transformed cells, the library size as determined by thecolony forming units (CFU) was estimated to be about 3.8×10⁶.

The transformed cells were cultured in LB media overnight, and on thenext day, protein expression was induced withIsopropyl-beta-D-thiogalactoside (IPDG). On the following day, theemulsion PCR was performed using the oil/surfactant mixtures containingMineral oil, non-ionic emulsifier (e.g., polaxamer 124, polaxamer 181,or ABIL® EM 90), and a surfactant (e.g., Triton X-100). This mixture wasadded to the ePCR master mixes, containing a buffer, CSR primeroligonucleotides, BSA, dNTPs, and 1×10⁸ cells. The emulsion was madeusing a TissueLyser (Qiagen®).

The emulsion PCR program was designed according to the selectivepressure used, modulating the number and type of primers for each round.The product from the emulsion PCR was extracted from emulsion using themethod known in the art, for example described by Williams et al. NatMethods. 2006 July; 3(7):545-50. A recovery PCR reaction is performedusing the product from the ePCR as a template. New primers and PCRprograms are designed for this purpose. After that, a third PCR reactionis performed using the Recovery PCR's product as a template. The productfrom the Re-Amp PCR is then cloned into a commercial vector (pET21b+)via Gibson Assembly, and an additional selection round occurs.

To promote strand displacement, the selective pressure applied includedi) progressively increasing the amount of double stranded regions withina nucleic acid template (i.e., increasing the amount of blockingoligos), and ii) decreasing the temperature to promote dsDNA formation.CSR primers would anneal to the template and after a few base pairs, theenzyme encountered one or more blocking primer(s), i.e., anoligonucleotide complementary to a region downstream of the CSR primers.Only the enzyme capable of displacing the double-stranded region wouldbe able to replicate its own encoding gene, and therefore would beenriched in the library. A total of 6 rounds of selective pressure wereperformed, as described in Table 1 below.

TABLE 1 Selective pressure on the finger and palm subdomains. Theconditions were progressively modulated to decrease the reactiontemperature and progressively increase the amount of double strandedregions within a nucleic acid template (i.e., the quantity of blockingoligos). Round Number Selective Pressure Round 1 Amplification at 72° C.Round 2 Amplification at 68° C. Round 3 Amplification at 66° C. and 2Xconcentration of blocking oligos Round 4 Amplification at 63° C. and 2Xconcentration blocking oligos Round 5 Amplification at 60° C. and 2Xconcentration of blocking oligos Round 6 Amplification at 60° C. and 4Xconcentration of blocking oligos

The primers were designed to have a sequence that is the complement of aregion of template/target DNA to which the primer hybridizes. A numberof primer design tools exist, for example PrimerSelect (Plasterer TN.PRIMERSELECT. Primer and probe design, Methods Mol. Biol., 1997, vol. 70(pg. 291-302)), Primer Express (Applied BiosystemsPrimer Express®Software Version 3.0 Getting Started Guide, 2004), OLIGO 7 (Rychlik W.OLIGO 7 primer analysis software, Methods Mol. Biol., 2007, vol. 402(pg. 35-60)) and Primer3 (Untergasser, A. et al. Primer3-newcapabilities and interfaces. Nucleic Acids Res 40, e115 (2012)). CSRprimers contain a non-complimentary region at the 5′ end (“tag”) so thatthe product can be extracted and enriched via PCR. The tags are used toprevent the carry-over and accumulation of amplifications resulting frombackground amplifications (amplification of DNA polymerase sequencesthat were not selected for in the ePCR step but were carried over asparental plasmid DNA to recovery PCR).

Blocking oligos were designed to bind downstream of the CSR primerbinding region, and had the 3′ end blocked with a C3 spacer to preventextension from the 3′ end. Primers were designed taking intoconsideration the necessary Melting Temperatures, since one of theselective pressures was reduced extension temperature. Calculating themelting temperature and performing thermodynamic modelling forestimating the propensity of primers to hybridize with other primers orto hybridize at unintended sites in the template offer an accurateapproach for predicting the energetic stability of DNA structures. TheTm is the temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium).

The sequencing data from each mutant was analyzed to identify mutationsin the nucleotide level, which were then translated to amino acids. Theamino acid mutations calculated frequency of each mutation per round wasobtained. The CSR Library was narrowed over the rounds of selection andshows many enriched mutations that are involved in strand-displacement.After each round of selection, the sequence of the enzyme was obtainedto elucidate which mutations are responsible for the strand-displacementactivity. Table 2 provides an overview of some of the mutations withinthe finger and palm subdomain responsible for increased stranddisplacing activity. Using the CSR techniques, novel mutations in thefinger/palm subdomains in a DNA polymerase were found. For example, themutations identified in over 30% of the mutant enzymes are identified inTable 2.

TABLE 2 Summary of point mutations identified in strand-displacingmutants; the point mutations are relative to SEQ ID NO: 1. Pointmutation F588L F588I Q520H E580G K465E A491V K472E

Example 2. Broader Mutational Analysis

Simultaneously, we extended the mutational analysis to the entire enzymeand not limiting it to a particular subdomain (e.g., the finger or palmsubdomain). Prior to performing CSR, an error prone PCR library wasgenerated with a target error rate of 8 to 15 mutations per clone usinga mutant sequencing enzyme having the sequence of SEQ ID NO:1. Thelibrary was cloned using pET21b+ following standard restriction enzymedigestion and ligation as well as a commercial vector pD431-SR from ATUMvia Gibson Assembly, using the Gibson Assembly® Master Mix (NEB CatalogNumber E2611S), as described above.

Both libraries were subjected to 10 rounds of selective pressure,described in Table 3, wherein each round decreased the reactiontemperature and/or increased the concentration of blocking oligos.

TABLE 3 Description of the selective pressure dimensions utilized todevelop strand-displacing variants. Selection Pressure DenaturationExtension Extension Excess Round # Tr temperature time blocking oligosRound 1 91° C. 72° C. 3 min 0 Round 2 91° C. 72° C. 3 min 0 Round 3 91°C. 68° C. 3 min 0 Round 4 91° C. 64° C. 3 min 2X Round 5 91° C. 60° C. 3min 2X Round 6 91° C. 60° C. 3 min 4X Round 7 91° C. 57° C. 3 min 4XRound 8 91° C. 55° C. 2 min 4X Round 9 96° C. 55° C. 1 min 4X Round 1098° C. 55° C. 30 seconds 4X

An additional selective pressure was applied in later rounds, decreasingthe reaction time from 3 minutes to a total extension time of 30seconds, to provide enzymes with strand-displacing activity andaccelerated processivity. A total of 195 unique point mutations wereobserved, however, the mutations occurring at the highest frequency areprovided in Table 4.

TABLE 4 Summary of some of the point mutations identified instrand-displacing mutants; the point mutations are relative to SEQ IDNO: 1. Point mutations Y7H A491V K13R R526H A40V E579G F75L F588L R97C,H T606I G149D G635D K192R V637D K199E N672D F241I H726D M275L V742AA316S F749Y K395E G765D K469T W769R K472N

Example 3. Strand Displacing Assay

Random clones from the library including one or more of thehigh-frequency mutations, as well as new variants based on the enrichedmutations, were screened for strand-displacement activity. The stranddisplacement assay was developed to measure the strand-displacingactivity of the mutants, as described in Harris et al. BioTechniques54:93-97 (February 2013), such that when extension occurs from the outerprimer, it displaces the extension strand produced from the inner primerby utilizing a polymerase that has strand displacement activity.Briefly, the assay includes annealing two outer primers, PrimA andPrimD, to a template nucleic acid. Two or more inner primers areannealed to the template, Prim B and PrimC, wherein the primershybridize to complementary regions between the outer primers. Nostrand-displacement activity is required to amplify from the innerprimers (e.g., generating small fragments from PrimB to PrimC). Variantspossessing strand-displacing activity are capable of generatingamplification products from the outer primers and the inner primers. Theresults of the strand-displacement assay are provided in Table 5.

TABLE 5 Results of the strand displacing assay. A ‘+’ is indicative ofstrand-displacing activity detection, whereas a ‘−‘ indicates nostrand-displacing activity was detected. The point mutations providedbelow are relative to SEQ ID NO: 1. SD Internal products Ref No.detected Mutations AMP1 + Q91H; R97H; D215G; G245S; D246E; G350S; R467C;K469T; A516S; E579G; F588L; H726D; V742A AMP2 + Y7H; K13R; Q91H; R97H;D215G; G245S; D246E; G350S; R467C; K469T; E579G; F588L; H634Y; H726D;V742A AMP3* + Y7H; N132S; E148G; I158V; D215E; K240M; G323S; E383G;K395R; G448S; K469E; K478R; F583L; E600K; G635D; E638D; A676V; R690H;R706H; V742A; Q759; G765D; W769R; K773E; G776D AMP4* + Y7H; K13R; A56V;I65V; R97C; E182D; S186C; R193H; P217S; Y279C; L333M; R364C; F446S;R526H; Y580C; F588L; T606I; A630G; H726D; W758; W769 AMP5* + K52R; V62G;I65V; R97C; E148D; T208S; K289R; I295N; E314D; R426H; K466Q; R483C;A491V; Q520H; R526H; R527H; G550V; F588L; T591A; 1611V; K642E; V644M;H726D; V742A; K752R; G767D; W769; I770M AMP6 + N23Y; F26S; D45N; V93I;R97H; E133V; I160V; A168T; A316P; R324H; S347N; K395E; L397M; Q462L;V514I; F588L; G635V; N636D; V656I; H726D; R753S; W769L AMP7* Y7H; D11G;H59L; R97H; F116L; M129T; E148D; K154E; F214I; I282T; G302D; K395E;E430Q; D566Y; E579G; F588L; V637D; H726D; V742A; W769R; K774 AMP8 +E25V; I71N; R97H; G155D; S247I; A331D; K395E; K469N; K478M; L544P;R559H; E579G; F588L; T623I; H726D; V742A; K774E AMP9 + Y7H; K13R; F75L;R97C; A117T; K229T; F283S; T319S; E579G; F588L; N653T; K693N; V742A;K762E; W769L; G776S AMP10 + Y7H; I8V; N23D; V66A; F75L; R97C; P217Q;F283S; K297E; T319S; T349A; R526H; E579G; F588L; M688V; H726D; Q737R;V742A AMP11 + Y7H; K13R; I38F; A40V; D98N; I109V; D141G; G149D; A168P;I176V; K192R; K199E; F214I; P286Q; A292T; R307S; S348N; K465N; C507S;R526H; G550D; F588L; T606I; E655D; R706C; V742A; W769R AMP12 + I51V;E67G; R97G; P104S; F116L; A139V; I142F; I207V; F261S; A292V; K465N;Q484L; E579G; F588L; T623A; K660Q; H726D; V742A; K752E; W769R AMP13* +E22K; K52R; I65V; Q57H; R97C; K174E; D215G; G245S; D246E; G350S; R467C;K469T; E579G; F588L; H726D; V742A; W758 AMP14 + Y7H; K13R; I65V; R97C;D98E; A117T; M190V; K289T; K317E; K391T; E579G; F588L; V742A; K771EAMP15 + E25K; R97C; A168T; R255H; F326Y; K478R; R526H; E556K; K558N;P573S; E581N; F588L; E600K; E601K; R686H; R724H; V742A; K752E; K762NAMP16 + Y7H; K13I; F75L; R97L; I160V; V170I; A316P; K469E; A491V; E579G;F588L; V637D; H726D; V742A AMP17* + Y7H; K13R; E50D; F75L; R97C; F214I;Y320F; F326Y; E378V; E427D; K463N; I464V; R532H; V551I; F588L; E665K;R690H; Y751H; L756 AMP18 + Y7H; K13R; I38F; A40V; D49E; F75I; I109V;D141G; G149D; A168P; I176V; K192R; K199E; F214I; K289; A292T; R307S;S348N; K465N; C507S; K558R; F588L; G635D; Y664; I667L; A730E; V742A;Q763; G765D; K773R AMP19 + Y7H; K13R; A40V; R97H; F116L; A117S; K154E;A281T; I415V; K477I; K552N; N569S; E577K; A585G; F588I; E655D; V742AAMP20 + Y7F; A40V; I109V; D141G; G149D; A168P; I176V; K192R; K199E;F214I; P239L; T272A; A292T; R307S; S348N; K465N; C507S; E579G; F588L;V742A; K773E; K774M AMP21 + Y7H; K13R; I38F; A40V; R97C; D141A; P286L;G350S; R467C; K469T; E579G; F588L; F721Y; H726D; V742A; L756C; R757A;W758G; Q759R; T761P; K762N; Q763R; V764L; G765V AMP22 + Y7H; I18V; R97C;A168S; F214S; G284S; P286Q; A292V; G304D; K391I; E431D; K477I; Y567H;E579G; F588L; H726D AMP23 + Y7H; K13R; V63I; F75L; R97C; E130D; F214S;F326Y; K391I; K478R; E618G; R706C; H726Y; I745L; F749Y AMP24 + V63A;F75L; R97C; D98G; R188H; F214I; G245S; D246E; T319I; G350S; E579G;F588L; R690H; H726D; V742A; K760N AMP25 + Y7H; I8V; N23D; V66A; F75L;R97C; P217Q; A298S; A316P; K465E; R526H; E577K; E601D; T606I; E655D;R706C; V742A; W769S AMP26 + E29D; Y30F; R97H; I160V; K229E; A511V;I548V; F588L; G635D; V742A AMP27 + Y7Q; I38F; A40V; Y86H; R97C; F261I;A296T; V434I; K437N; K465E; R526H; A561S; N569K; F588L; T606I; V644M;N672D; K693I; V742A; W758; W769R AMP28 + K13R; R32H; F75L; R97C; K201I;Y209H; I256V; Y291H; E383D; G400D; R526C; E579G; F588L; E638G; A730VAMP29 + F75L; R97C; I142T; V278I; A281T; A292E; M329L; P372S; H440N;E563G; E579G; F588L; D729Y; V742A AMP30* + Y7H; R97L; I176F; L275P;Y279F; Q332P; V438I; L479Q; K535R; R560H; D566G; F588L; T591A; V644M;A676V; H726D; L756 AMP31 + Y7H; K13R; I38F; A40V; I65T; V93I; I109V;G149D; A168P; I176V; K192R; K199E; F214I; A292T; R307S; S348N; K465N;C507S; F588L; G635D; K660E; A676V; R690H; R706H; V742A; Q759; G765D;G767V; W769R; G776D AMP32 + Y7H; K13R; I51V; E67G; I96F; R97S; F110L;K154M; I207V; K289N; E386D; E430D; K469T; S471L; R560H; F588L; T591A;W616C; V644M; A676V; H726D; L756 AMP33 + I65V; R97H; F283S; V308M;K465E; F588L; T591A; I604M; G635D; K727T AMP34 + Y7H; K13R; L76P; K192E;K289T; R364C; L397M; Q484H; E563G; F588L; V637D; R706H; V742A; L766PAMP35 + Y7H AMP36 + K13R AMP37 + Q91H AMP38 + R97H AMP39 + R97C AMP40 +D215G AMP41 + D215E AMP42 + G245S AMP43 D246E AMP44 G350S AMP45 R467CAMP46 K469T AMP47 − R467C & K469T AMP48 + E579G AMP49 + F588L AMP50 +E579G & F588L AMP51 + H634Y AMP52 + H726D AMP53 V742A AMP54 + N23YAMP55 + F26S AMP56 − V742A/F588L AMP57 + Y7H/R97H AMP58 + Y7H/R97C AMP59V742A/H726D AMP60 W769R AMP61 R753S AMP62 D45N AMP63 E133V AMP64R97H/V742A/F588L AMP65 Y7H/V742A/F588L AMP66 E579G/V742A/F588L AMP67H726D/V742A/F588L AMP68 W769L

Clones from the library including one or more of the high-frequencymutations, as well as new variants based on the enriched mutations, werescreened for strand-displacement activity and the point mutationsproviding increased strand-displacing activity (increased relative to acontrol, SEQ ID NO:1) are provided in Table 5. Some of the proteins weresmaller and displayed different thermal stability profile than thecontrol; investigating the amino acid sequence and the encodingpolynucleotide sequence identifies one or more stop codons. A stop codonis a nucleotide triplet that signals the termination of the translationprocess of the current polypeptide. The smaller enzymes are indicated inTable 5 by a identifier, and the corresponding bold amino acid (e.g.,L756 of AMP 17) is associated with the stop codon.

SEQUENCESAmino Acid Sequence of wild type P. horikoshii OT3 (SEQ ID NO: 1):MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLRDDSAIDEIKKITAQRHGKVVRIVETEKIQRKFLGRPIEVWKLYLEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEGNEKLTFLAVDIETLYHEGEEFGKGPVIMISYADEEGAKVITWKKIDLPYVEVVSSEREMIKRLIRVIKEKDPDVIITYNGDNFDFPYLLKRAEKLGIKLLLGRDNSEPKMQKMGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWEGCEEYDVAPKVGHRFCKDFPGFIPSLLGQLLEERQKIKKRMKESKDPVEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGROYIDLVRRELEARGFKVLYIDTDGLYATIPGVKDWEEVKRRALEFVDYINSKLPGVLELEYEGFYARGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVKIVKDVTEKLTNYEVPPEKLVIYEQITRPINEYKAIGPHVAVAKRLMARGIKVKPGMVIGYIVLRGDGPISKRAISIEEFDPRKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWIKVKKSDNA Sequence of wild type P. horikoshii OT3 (SEQ ID NO: 2):Pyrococcus horikoshii DNA Polymerase geneATGATTCTGGACGCTGATTATATTACTGAAGATGGTAAACCGATTATTCGTATTTTTAAAAAAGAAAATGGCGAGTTCAAAGTTGAATATGACCGTAACTTTCGTCCGTACATCTACGCGCTGTTGCGCGACGATAGCGCGATCGATGAGATTAAGAAAATTACCGCGCAGCGTCATGGTAAAGTTGTTCGCATCGTTGAAACCGAGAAAATTCAACGTAAATTCCTGGGCCGCCCAATTGAAGTGTGGAAGCTGTACCTGGAGCATCCGCAAGATGTCCCGGCGATCCGTGACAAGATTCGCGAGCACCCGGCCGTCGTCGACATTTTCGAATACGATATTCCGTTCGCAAAGCGTTACCTGATCGATAAGGGTCTGACCCCGATGGAGGGTAATGAAAAGCTGACGTTCCTGGCTGTCGATATTGAAACGTTGTACCACGAGGGTGAAGAGTTTGGTAAGGGCCCGGTCATTATGATCAGCTACGCGGATGAAGAGGGCGCCAAAGTTATCACGTGGAAAAAAATTGATCTGCCGTACGTTGAAGTTGTGTCCAGCGAGCGCGAGATGATTAAACGCTTGATTCGTGTGATTAAAGAAAAAGATCCAGACGTGATCATTACCTATAATGGTGACAACTTTGACTTTCCGTACTTGCTGAAACGTGCTGAGAAACTGGGTATCAAGCTGTTGCTGGGTCGCGATAATAGCGAGCCGAAGATGCAAAAAATGGGCGATAGCCTGGCAGTCGAGATCAAGGGTCGCATCCACTTTGATCTCTTTCCGGTGATTCGTCGCACGATCAATCTGCCGACCTATACGCTGGAAGCTGTCTACGAGGCAATCTTTGGTAAGCCGAAAGAAAAAGTCTATGCGGACGAAATTGCGAAAGCGTGGGAAACCGGCGAGGGCCTGGAGCGTGTGGCAAAGTACTCTATGGAAGATGCCAAAGTGACCTATGAACTGGGTCGTGAGTTCTTCCCAATGGAAGCCCAGTTGGCGCGCTTGGTGGGCCAACCGGTTTGGGACGTTTCCCGTAGCAGCACCGGTAACCTGGTTGAGTGGTTTCTGTTGCGTAAAGCGTATGAGCGTAATGAACTGGCACCGAACAAGCCTGACGAGAAAGAATATGAACGTCGCCTGCGTGAATCTTACGAGGGTGGTTACGTCAAAGAACCGGAAAAGGGTCTGTGGGAAGGCATCGTGAGCCTGGATTTCCGTAGCCTGTACCCTAGCATCATCATCACGCACAATGTTAGCCCGGACACCCTGAACCGCGAGGGCTGCGAAGAGTACGACGTTGCGCCGAAAGTCGGCCATCGTTTTTGTAAAGACTTCCCTGGTTTCATCCCAAGCCTGCTGGGTCAGCTGCTGGAAGAGAGACAGAAAATTAAAAAACGCATGAAAGAATCGAAAGATCCGGTTGAGAAAAAGCTGCTGGATTACCGCCAGCGTGCCATCAAGATTCTGGCTAACTCATATTATGGCTACTACGGTTATGCTAAAGCGCGTTGGTACTGTAAAGAGTGCGCGGAGTCCGTCACCGCGTGGGGTCGCCAGTATATCGATCTGGTGCGTCGCGAGCTGGAAGCGCGTGGTTTTAAGGTCCTGTACATCGATACTGACGGTCTGTATGCAACCATCCCTGGTGTCAAAGACTGGGAAGAGGTTAAGCGTCGTGCACTGGAATTTGTGGACTATATCAATTCTAAGTTGCCGGGTGTGCTGGAGCTGGAGTACGAAGGCTTCTATGCACGCGGCTTTTTCGTTACGAAAAAGAAATACGCACTGATCGACGAAGAGGGCAAGATTGTGACTCGTGGTCTGGAAATCGTTCGTCGCGACTGGAGCGAGATTGCAAAAGAAACCCAAGCTCGCGTTCTGGAAGCAATCCTGAAACATGGTAACGTCGAAGAAGCCGTCAAGATCGTGAAAGATGTCACCGAAAAGTTGACCAACTACGAAGTTCCACCGGAAAAACTGGTGATTTATGAGCAAATCACGCGTCCGATCAATGAATATAAGGCCATTGGCCCGCACGTCGCGGTGGCCAAGCGCCTGATGGCGCGTGGTATCAAAGTGAAACCGGGTATGGTTATTGGTTACATCGTGCTGCGTGGCGACGGCCCGATTAGCAAACGTGCGATCAGCATTGAAGAATTTGACCCGCGTAAGCACAAATATGACGCGGAATACTATATCGAGAATCAAGTGCTGCCGGCCGTGGAACGCATTCTGAAAGCTTTCGGCTACAAGCGTGAAGATTTGCGCTGGCAGAAAACCAAACAGGTTGGTCTTGGTGCGTGGATCAAGGTCAAAAAGTCCTAA Pyrococcus abyssi (SEQ ID NO: 3)MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKDDSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYLEHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKSIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKSKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTFELGKEFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNRENCKEYDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKDPVEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIKEKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLAAKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAEYYIENQVLPAVERILRAFGYRKEDLKYQKTKQVGLGAWLKF Pyrococcus woesei (SEQ ID NO: 4)MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSPyrococcus furiosus (SEQ ID NO: 5)MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS Pyrococcus glycovorans (SEQ ID NO: 6)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVKKITAERHGKIVRIVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGPDTLNREGCMEYDVAPEVKHKFCKDFPGFIPSLLKRLLDERQEIKRRMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKRKALEFVEYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDPRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNVKKK Pyrococcus sp. NA2 (SEQ ID NO: 7)MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS Pyrococcus sp. ST700 (SEQ ID NO: 8)MILDADYITENGKPIIRLFKKENGKFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVSKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELSFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLVRIIREKDPDIIITYNGDNFDFPYLLKRAEKLGIKLPLGRDNSEPKMQRLGESLAVEIKGRIHFDLFPVIRRTINLPTYTLRTVYEAIFGKPKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKEYEKRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNREGCGKYDEAPEVGHRFCKDFPGFIPSLLGDLLEERQKIKKRMKESKDPIEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRKALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVLLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKKEDLRWQPyrococcus kukulkanii (SEQ ID NO: 9)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVRKITAERHGKIVRIIDAEKVRKKFLGRPIEVWRLYFEHPQDVPAIRDKIREHSAVIDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCREYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQEIKRKMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKKKALEFVDYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNIKKK Pyrococcus yayanosii (SEQ ID NO: 10)MILDADYITENGKPVVRIFKKENGEFKVEYDRSFRPYIYALLRDDSAIEDIKKITAERHGKVVRVVEAEKVRKKFLGRPIEVWKLYFEHPQDVPAIREKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGDEFGSGPIIMISYADEKGAKVITWKGVDLPYVEVVSSEREMIKRFLRVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPIGRDGSEPKMQRMGDGFAVEVKGRIHFDIYPVIRRTINLPTYTLEAVYEAVFGRPKEKVYPNEIARAWENCKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNRPDEREYERRLRESYEGGYVKEPEKGLWEGIIYLDFRSLYPSIIITHNISPDTLNKEGCNSYDVAPKVGHRFCKDFPGFIPSLLGQLLDERQKIKRKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVSRELEKRGFKVLYIDTDGLYATIPGSREWDKIKERALEFVKYINARLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKEGNLEKAVKIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKAVGPHVAVAKRLAARGIKVRPGMVIGYLVLRGDGPISRRAIPAEEFDPSRHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRYQKTRQAGLDAWLKRKASL Pyrococcus sp. ST04 (SEQ ID NO: 11)MILDADYITEDGKPVIRLFKKENGEFKIEYDRTFKPYIYALLKDDSAIDEVRKVTAERHGKIVRIIDVEKVKKKYLGRPIEVWKLYFEHPQDVPAIREKIREHPAVVEIFEYDIPFAKRYLIDKGIVPMDGDEELKLLAFDIETLYHEGEEFGKGPILMISYADEEGAKVITWKRINLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDFPYLVKRAEKLGIKLPLGRDGSPPKMQRLGDMNAVEIKGRIHFDLYHVVRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAQVTYELGKEFFPMEVQLTRLVGQPLYPSIIITHNVSPDTLNREGCRKYDIAPEVGHKFCKDVEGFIPSLLGHLLEERQKIKRKMKATINPVEKKLLDYRQKAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVRKELEGKFGFKVLYIDTDGLYATIPRGDPAEIKKKALEFVRYINEKLPGLLELEYEGFYRRGFFVTKKKYALIDEEDKIITRGLEIVRRDWSEIAKETQAKVLEAILKEGNVEKAVKIVKEVTEKLMKYEVPPEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGVKVRPGMVIGYIVLRGDGPISKRAILAEEYDPRKNKYDAEYYIENQVLPAVLRILEAFGYKKEDLKYQKSRQVGLGAWIKVKK Pyrococcus sp. GB-D (SEQ ID NO: 12)MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVRKITAERHGKIVRIIDAEKVRKKFLGRPIEVWRLYFEHPQDVPAIRDKIREHSAVIDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGPDTLNREGCREYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQEIKRKMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKKKALEFVDYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDLRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNIKKK

EMBODIMENTS

Embodiment 1. A polymerase comprising an amino acid sequence that is atleast 80% identical to a continuous 500 amino acid sequence within SEQID NO: 1; comprising a first mutation at amino acid position 7 or anamino acid position corresponding to position 7, wherein the mutation ishistidine, lysine, or arginine; and a second mutation at amino acidposition 588 or an amino acid position corresponding to position 588;wherein the second mutation comprises leucine, isoleucine, valine,alanine, or glycine.

Embodiment 2. The polymerase of Embodiment 1, wherein the first mutationis histidine.

Embodiment 3. The polymerase of Embodiments 1 or 2, comprising amutation at amino acid position 76 or an amino acid positioncorresponding to amino acid position 76, wherein the mutation isproline, histidine, arginine, or glycine.

Embodiment 4. The polymerase of any one of Embodiments 1 to 3,comprising a mutation at amino acid position 76 or an amino acidposition corresponding to amino acid position 76, wherein the mutationis proline, histidine, or glycine.

5. The polymerase of any one of Embodiments 1 to 4, comprising amutation at amino acid position 742 or an amino acid positioncorresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.

Embodiment 6. The polymerase of any one of Embodiments 1 to 4,comprising a mutation at amino acid position 742 or an amino acidposition corresponding to position 742, wherein the mutation is alanineor glycine.

Embodiment 7. The polymerase of any one of Embodiments 1 to 6,comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginineor histidine.

Embodiment 8. The polymerase of any one of Embodiments 1 to 6,comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine.

Embodiment 9. The polymerase of any one of Embodiments 1 to 8,comprising a mutation at amino acid position 397 or an amino acidposition corresponding to position 397, wherein the mutation ismethionine, cysteine, isoleucine, valine, or alanine.

Embodiment 10. The polymerase of any one of Embodiments 1 to 8,comprising a mutation at amino acid position 397 or an amino acidposition corresponding to position 397, wherein the mutation ismethionine, valine, or alanine.

Embodiment 11. A polymerase comprising an amino acid sequence that is atleast 80% identical to a continuous 500 amino acid sequence within SEQID NO: 1; comprising a first mutation at amino acid position 97 or anamino acid position corresponding to position 97, wherein the firstmutation comprises cysteine, histidine, lysine, serine, threonine, ormethionine; and a second mutation at amino acid position 588 or an aminoacid position corresponding to position 588; wherein the second mutationcomprises leucine, isoleucine, valine, alanine, or glycine.

Embodiment 12. The polymerase of Embodiment 1, wherein the firstmutation is cysteine, histidine, or serine.

Embodiment 13. The polymerase of any one of Embodiments 1 to 12, whereinthe second mutation is leucine, isoleucine, valine, or alanine.

Embodiment 14. The polymerase of Embodiment any one of Embodiments 11 to13, comprising a mutation at amino acid position 742 or an amino acidposition corresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.

Embodiment 15. The polymerase of Embodiment any one of Embodiments 11 to13, comprising a mutation at amino acid position 742 or an amino acidposition corresponding to position 742, wherein the mutation is alanineor glycine.

Embodiment 16. The polymerase of Embodiment any one of Embodiments 1 to15, comprising a mutation at amino acid position 726 or an amino acidposition corresponding to amino acid position 726, wherein the mutationis aspartic acid, glutamic acid, asparagine, or glutamine.

Embodiment 17. The polymerase of Embodiment any one of Embodiments 1 to16, comprising a mutation at amino acid position 769 or an amino acidposition corresponding to position 769, wherein the mutation is leucine,isoleucine, valine, alanine, or glycine.

Embodiment 18. The polymerase of Embodiment any one of Embodiments 11 to17, comprising a mutation at amino acid position 7 or an amino acidposition corresponding to position 7, wherein the mutation is histidine,lysine, or arginine.

Embodiment 19. The polymerase of Embodiment any one of Embodiments 1 to18, comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine,leucine, isoleucine, or histidine.

Embodiment 20. The polymerase of Embodiment any one of Embodiments 1 to19, comprising a mutation at amino acid position 241 or an amino acidposition corresponding to position 241, wherein the mutation is leucine,isoleucine, alanine, valine, or glycine.

Embodiment 21. The polymerase of Embodiment any one of Embodiments 1 to20, comprising a mutation at amino acid position 472 or an amino acidposition corresponding to position 472, wherein the mutation isasparagine, aspartic acid, glutamic acid, or glutamine.

Embodiment 22. The polymerase of Embodiment any one of Embodiments 1 to20, comprising a mutation at amino acid position 472 or an amino acidposition corresponding to position 472, wherein the mutation isasparagine or glutamic acid.

Embodiment 23. The polymerase of Embodiment any one of Embodiments 1 to22, comprising a mutation at amino acid position 469 or an amino acidposition corresponding to position 469, wherein the mutation isthreonine, serine, cysteine, or methionine.

Embodiment 24. The polymerase of Embodiment any one of Embodiments 1 to22, comprising a mutation at amino acid position 469 or an amino acidposition corresponding to position 469, wherein the mutation isasparagine, aspartic acid, glutamic acid, or glutamine.

Embodiment 25. The polymerase of Embodiment any one of Embodiments 1 to24, comprising: a mutation at amino acid position 520 or an amino acidposition corresponding to position 520, wherein the mutation ishistidine, lysine, or arginine; a mutation at amino acid position 465 oran amino acid position corresponding to position 465, wherein themutation is asparagine, aspartic acid, glutamic acid, or glutamine;and/or a mutation at amino acid position 491 or an amino acid positioncorresponding to position 491, wherein the mutation is glycine, valine,leucine, or isoleucine.

Embodiment 26. The polymerase of Embodiment any one of Embodiments 1 to25, comprising a glutamine, valine, arginine, or alanine at amino acidposition 93 or an amino acid position corresponding to position 93.

Embodiment 27. The polymerase of Embodiment any one of Embodiments 1 to26, comprising an alanine at amino acid position 141 or an amino acidposition corresponding to position 141; and an alanine at amino acidposition 143 or an amino acid position corresponding to position 143.

Embodiment 28. A method of incorporating a nucleotide into a nucleicacid sequence comprising combining in a reaction vessel: (i) a nucleicacid template, (ii) a nucleotide solution, and (iii) a polymerase,wherein the polymerase is a polymerase of Embodiment any one ofEmbodiments 1 to 27.

Embodiment 29. A method of amplifying a nucleic acid sequencecomprising: a. hybridizing a nucleic acid template with a primer to forma primer-template hybridization complex; b contacting theprimer-template hybridization complex with a DNA polymerase and aplurality of nucleotides, wherein the DNA polymerase is the polymeraseof Embodiment any one of Embodiments 1 to 27; c incorporating one ormore nucleotides into the primer-template hybridization complex with theDNA polymerase to generate amplification products, thereby amplifying anucleic acid sequence.

Embodiment 30. A kit comprising a polymerases of any one of claims 1 to28.

Embodiment 31. A polymerase comprising an amino acid sequence that is atleast 80% identical to a continuous 500 amino acid sequence within SEQID NO: 1; comprising a first mutation at amino acid position 588 or anamino acid position corresponding to position 588; wherein the firstmutation is leucine, isoleucine, valine, alanine, or glycine; and asecond mutation at amino acid position 7 or an amino acid positioncorresponding to position 7, wherein the mutation is histidine, lysine,or arginine; at amino acid position 97 or an amino acid positioncorresponding to position 97, wherein the mutation is cysteine,histidine, lysine, serine, threonine, or methionine; or at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is leucine, isoleucine, alanine, or glycine.

Embodiment 32. The polymerase of Embodiment 31, wherein the secondmutation is a mutation at amino acid position 7 or an amino acidposition corresponding to position 7, wherein the mutation is histidine,lysine, or arginine.

Embodiment 33. The polymerase of Embodiment 32, further comprising amutation at amino acid position 742 or an amino acid positioncorresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.

Embodiment 34. The polymerase of Embodiment 31, wherein the secondmutation is a mutation at amino acid position 97 or an amino acidposition corresponding to position 97, wherein the mutation is cysteine,histidine, lysine, serine, threonine, or methionine.

Embodiment 35. The polymerase of Embodiment 34, further comprising amutation at amino acid position 742 or an amino acid positioncorresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.

Embodiment 36. The polymerase of Embodiment 31, wherein the secondmutation is a mutation at amino acid position 742 or an amino acidposition corresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.

Embodiment 37. The polymerase of any one of Embodiments 31 to 36,comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine,isoleucine, methionine, or histidine.

Embodiment 38. The polymerase of Embodiment 31, wherein the secondmutation is a mutation at amino acid position 97 or an amino acidposition corresponding to position 97, wherein the mutation is cysteineor histidine.

Embodiment 39. The polymerase of any one of Embodiments 31 to 38,wherein the first mutation is leucine, isoleucine, valine, or alanine.

Embodiment 40. The polymerase of any one of Embodiments 31 to 38,wherein the first mutation is leucine or valine.

Embodiment 41. The polymerase of any one of Embodiments 31 to 40,comprising a mutation at amino acid position 579 or an amino acidposition corresponding to position 579, wherein the mutation is alanineor glycine.

Embodiment 42. The polymerase of any one of Embodiments 31 to 41,comprising a mutation at amino acid position 726 or an amino acidposition corresponding to amino acid position 726, wherein the mutationis aspartic acid, glutamic acid, asparagine, or glutamine.

Embodiment 43. The polymerase of any one of Embodiments 31 to 42,comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine,leucine, isoleucine, or histidine.

Embodiment 44. The polymerase of any one of Embodiments 31 to 43,comprising: a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine,leucine, isoleucine, or histidine; and a mutation at amino acid position579 or an amino acid position corresponding to position 579, wherein themutation is alanine or glycine.

Embodiment 45. The polymerase of Embodiment 31, comprising: R97H, F588L,G635D, and V742A; K13R, R97C, E579G, and F588L; R97C, E563G, E579G,F588L, and V742A; R97H, F588L, and G635D; R97C, F588L, and V742A; Y7H,K13I, R97L, E579G, F588L, and V742A; Y7H, K13R, R97H, and V742A; 7H,K13R, R97C, D141A, E579G, F588L, and V742A; R97C, E579G, F588L, andV742A; Y7H, R97C, E579G, and F588L; Y7H, R97C, and V742A; or Y7H, K13R,E563G, F588L, and V742A. Embodiment 46. The polymerase of Embodiment 31,comprising: E29D, Y30F, R97H, I160V, K229E, A511V, I548V, F588L, G635D,and V742A; K13R, R32H, F75L, R97C, K201I, Y209H, I256V, Y291H, E383D,G400D, R526C, E579G, F588L, E638G, and A730V; F75L, R97C, I142T, V278I,A281T, A292E, M329L, P372S, H440N, E563G, E579G, F588L, D729Y, andV742A; I65V, R97H, F283S, V308M, K465E, F588L, T591A, I604M, G635D, andK727T; E25K, R97C, A168T, R255H, F326Y, K478R, R526H, E556K, K558N,P573S, E581N, F588L, E600K, E601K, R686H, R724H, V742A, K752E, andK762N; Y7H, K13I, F75L, R97L, I160V, V170I, A316P, K469E, A491V, E579G,F588L, V637D, H726D, and V742A; Y7H, K13R, A40V, R97H, F116L, A117S,K154E, A281T, I415V, K477I, K552N, N₅₆₉S, E577K, A585G, F588I, E655D,and V742A; Y7H, K13R, I38F, A40V, R97C, D141A, P286L, G350S, R467C, andK469T, E579G, F588L, F721Y, H726D, V742A, L756C, R757A, W758G, Q759R,T761P, K762N, Q763R, V764L, and G765V; V63A, F75L, R97C, D98G, R188H,F214I, G245S, D246E, T319I, G350S, E579G, F588L, R690H, H726D, V742A,and K760N; Y7H, 118V, R97C, A168S, F214S, G284S, P286Q, A292V, G304D,K391I, E431D, K477I, Y567H, E579G, F588L, and H726D; Y7H, I8V, N₂₃D,V66A, F75L, R97C, P217Q, A298S, A316P, K465E, R526H, E577K, E601D,T606I, E655D, R706C, V742A, and W769S; or Y7H, K13R, L76P, K192E, K289T,R364C, L397M, Q484H, E563G, F588L, V637D, R706H, V742A, and L766P.

Embodiment 47. The polymerase of any one of Embodiments 31 to 46,comprising an alanine at amino acid position 141 or an amino acidposition corresponding to position 141; and an alanine at amino acidposition 143 or an amino acid position corresponding to position 143.

Embodiment 48. A method of incorporating a nucleotide into a nucleicacid sequence comprising combining in a reaction vessel: (i) a nucleicacid template, (ii) a nucleotide solution, and (iii) a polymerase,wherein the polymerase is a polymerase of any one of Embodiments 31 to47.

Embodiment 49. A method of amplifying a nucleic acid sequencecomprising: hybridizing a nucleic acid template with a primer to form aprimer-template hybridization complex; b. contacting the primer-templatehybridization complex with a DNA polymerase and a plurality ofnucleotides, wherein the DNA polymerase is the polymerase of any one ofEmbodiments 31 to 47; c. incorporating one or more nucleotides into theprimer-template hybridization complex with the DNA polymerase togenerate amplification products, thereby amplifying a nucleic acidsequence.

Embodiment 50. A kit comprising the polymerase of any one of Embodiments31 to 47.

What is claimed is:
 1. A polymerase comprising an amino acid sequencethat is at least 80% identical to a continuous 500 amino acid sequencewithin SEQ ID NO: 1; comprising a first mutation at amino acid position588 or an amino acid position corresponding to position 588; wherein thefirst mutation is leucine, isoleucine, valine, alanine, or glycine; anda second mutation: at amino acid position 7 or an amino acid positioncorresponding to position 7, wherein the second mutation is histidine,lysine, or arginine; at amino acid position 97 or an amino acid positioncorresponding to position 97, wherein the second mutation is cysteine,histidine, lysine, serine, threonine, or methionine; or at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the second mutation is leucine, isoleucine, alanine, or glycine.2. The polymerase of claim 1, wherein the second mutation is a mutationat amino acid position 7 or an amino acid position corresponding toposition 7, wherein the mutation is histidine, lysine, or arginine. 3.The polymerase of claim 2, further comprising a mutation at amino acidposition 742 or an amino acid position corresponding to position 742,wherein the mutation is leucine, isoleucine, alanine, or glycine.
 4. Thepolymerase of claim 1, wherein the second mutation is a mutation atamino acid position 97 or an amino acid position corresponding toposition 97, wherein the mutation is cysteine, histidine, lysine,serine, threonine, or methionine.
 5. The polymerase of claim 4, furthercomprising a mutation at amino acid position 742 or an amino acidposition corresponding to position 742, wherein the mutation is leucine,isoleucine, alanine, or glycine.
 6. The polymerase of claim 1, whereinthe second mutation is a mutation at amino acid position 742 or an aminoacid position corresponding to position 742, wherein the mutation isleucine, isoleucine, alanine, or glycine.
 7. The polymerase of claim 1,comprising a mutation at amino acid position 13 or an amino acidposition corresponding to position 13, wherein the mutation is arginine,isoleucine, methionine, or histidine.
 8. The polymerase of claim 1,wherein the second mutation is a mutation at amino acid position 97 oran amino acid position corresponding to position 97, wherein themutation is cysteine or histidine.
 9. The polymerase of claim 1, whereinthe first mutation is leucine, isoleucine, valine, or alanine.
 10. Thepolymerase of claim 1, wherein the first mutation is leucine or valine.11. The polymerase of claim 1, comprising a mutation at amino acidposition 579 or an amino acid position corresponding to position 579,wherein the mutation is alanine or glycine.
 12. The polymerase of claim1, comprising a mutation at amino acid position 726 or an amino acidposition corresponding to amino acid position 726, wherein the mutationis aspartic acid, glutamic acid, asparagine, or glutamine.
 13. Thepolymerase of claim 1, comprising a mutation at amino acid position 13or an amino acid position corresponding to position 13, wherein themutation is arginine, leucine, isoleucine, or histidine.
 14. Thepolymerase of claim 1, comprising: a mutation at amino acid position 13or an amino acid position corresponding to position 13, wherein themutation is arginine, leucine, isoleucine, or histidine; and a mutationat amino acid position 579 or an amino acid position corresponding toposition 579, wherein the mutation is alanine or glycine.
 15. Thepolymerase of claim 1, comprising: R97H, F588L, G635D, and V742A; K13R,R97C, E579G, and F588L; R97C, E563G, E579G, F588L, and V742A; R97H,F588L, and G635D; R97C, F588L, and V742A; Y7H, K13I, R97L, E579G, F588L,and V742A; Y7H, K13R, R97H, and V742A; Y7H, K13R, R97C, D141A, E579G,F588L, and V742A; R97C, E579G, F588L, and V742A; Y7H, R97C, E579G, andF588L; Y7H, R97C, and V742A; or Y7H, K13R, E563G, F588L, and V742A. 16.The polymerase of claim 1, comprising: E29D, Y30F, R97H, I160V, K229E,A511V, I548V, F588L, G635D, and V742A; K13R, R32H, F75L, R97C, K201I,Y209H, I256V, Y291H, E383D, G400D, R526C, E579G, F588L, E638G, andA730V; F75L, R97C, I142T, V278I, A281T, A292E, M329L, P372S, H440N,E563G, E579G, F588L, D729Y, and V742A; I65V, R97H, F283S, V308M, K465E,F588L, T591A, I604M, G635D, and K727T; E25K, R97C, A168T, R255H, F326Y,K478R, R526H, E556K, K558N, P573S, E581N, F588L, E600K, E601K, R686H,R724H, V742A, K752E, and K762N; Y7H, K13I, F75L, R97L, I160V, V170I,A316P, K469E, A491V, E579G, F588L, V637D, H726D, and V742A; Y7H, K13R,A40V, R97H, F116L, A117S, K154E, A281T, I415V, K477I, K552N, N569S,E577K, A585G, F588I, E655D, and V742A; Y7H, K13R, I38F, A40V, R97C,D141A, P286L, G350S, R467C, and K469T, E579G, F588L, F721Y, H726D,V742A, L756C, R757A, W758G, Q759R, T761P, K762N, Q763R, V764L, andG765V; V63A, F75L, R97C, D98G, R188H, F214I, G245S, D246E, T319I, G350S,E579G, F588L, R690H, H726D, V742A, and K760N; Y7H, 118V, R97C, A168S,F214S, G284S, P286Q, A292V, G304D, K391I, E431D, K477I, Y567H, E579G,F588L, and H726D; Y7H, I8V, N23D, V66A, F75L, R97C, P217Q, A298S, A316P,K465E, R526H, E577K, E601D, T606I, E655D, R706C, V742A, and W769S; orY7H, K13R, L76P, K192E, K289T, R364C, L397M, Q484H, E563G, F588L, V637D,R706H, V742A, and L766P.
 17. The polymerase of claim 1, comprising analanine at amino acid position 141 or an amino acid positioncorresponding to position 141; and an alanine at amino acid position 143or an amino acid position corresponding to position
 143. 18. A method ofincorporating a nucleotide into a nucleic acid sequence comprisingcombining in a reaction vessel: (i) a nucleic acid template, (ii) anucleotide solution, and (iii) a polymerase, wherein the polymerase is apolymerase of claim
 1. 19. A method of amplifying a nucleic acidsequence comprising: a. hybridizing a nucleic acid template with aprimer to form a primer-template hybridization complex; b. contactingthe primer-template hybridization complex with a DNA polymerase and aplurality of nucleotides, wherein the DNA polymerase is the polymeraseof claim 1; c. incorporating one or more nucleotides into theprimer-template hybridization complex with the DNA polymerase togenerate amplification products, thereby amplifying a nucleic acidsequence.
 20. A kit comprising the polymerase of claim 1.